Field of the Invention

The present invention relates to genetically engineered microorganisms, such as bacteria, modified to increase production of cellulose and methods of producing said genetically engineered microorganisms.

Background

Drought and extreme heat are the largest climate-related threats to global agricultural production. A worsening climate means more extreme, unpredictable weather events that can range from acute large volumes of rain to prolonged periods of drought. These events have direct impact on crop yield. When rain occurs, the soil has a maximum absorption capacity, and the crop has a maximum uptake retention capacity. The remaining water, which can be substantial in dramatic weather events, is not utilised and often runs off the land, leading to localised flooding. Water shortage is a major global concern. 70% of global water consumption is from agricultural demand, and this is expect to increase 19% by 2050. Water consumption is a major impediment for global crop production. Within environments where drought is prevalent, low crop yields occur. By increasing the crop’s tolerance to environmental drought, it is possible to improve the sustainability of the global crop supply. Due to rain precipitation in hotter climates being more sporadic, farmers are forced to irrigate their land continuously. This requires huge volumes of water that will ultimately not be utilised by the plant and will evaporate. The abiotic stress incurred by crops in drought prone climates, results in reduced yield and inefficient water management. With a growing population and an ever worsening climate conditions, many strategies have been suggested to alleviate water demand, however as of yet, none have used a biological active water retentive solution to crop water management. Previous strategies have focused on genetically modifying the crop directly, to make them more resistant, or through the addition of additives in the soil. However, none of these approaches have provided an effective solution to the problem of water shortage in global crop production. Crop sustainability is essential for population growth and a food source for billions of people. Not only do crops feed the human population, they also support the animal farming industry, as a food supply. Therefore, now more than ever, a drastic change is required to support our growing world, but this solution must be environmentally friendly and efficient.

Cellulose

Cellulose is a polysaccharide consisting of a linear chain of several hundred to many thousands of b(1 4) linked D-glucose units. Cellulose is an important structural component of the primary cell wall of green plants, many forms of algae and the oomycetes. Additionally, some species of bacteria, principally of the genera Acetobacter, Sarcina ventriculi and Agrobacterium, secrete cellulose to firm biofilms. Bacterial cellulose is now produced for a variety of commercial applications including textiles, cosmetics, and food products, as well as medical applications. Expression of bacterial cellulose in bacteria has been described in the art. For example, Chinese patent application CN108060112 describes a bacterial cellulose producing bacterial strain, specifically, overexpression of a BcsB subunit in Acetobacter xylinum. Additionally, Buldum etal. (2018) describes recombinant biosynthesis of bacterial cellulose in genetically modified Escherichia coli. Whilst Florea etal. (2016) describes engineering control of bacterial cellulose production in Komagataeibacter rhaeticus using a genetic toolkit.

Carbon capture

Carbon sequestration on agricultural land is one way to reduce carbon emissions from agriculture and mitigate climate change. Atmospheric concentrations of carbon dioxide can be lowered either by reducing emissions or by taking carbon dioxide out of the atmosphere and storing it in the soil. The long-term conversion of grassland and forestland to cropland (and grazing lands) has resulted in historic losses of soil carbon worldwide but there is a major potential for increasing soil carbon through restoration of degraded soils and widespread adoption of soil conservation practices. The decline in soil quality has also been exacerbated by the use of chemical fertilisers.

Historically, land-use conversion and soil cultivation have been a prominent source of greenhouse gases (GHGs) to the atmosphere. It is estimated that they are still responsible for about one-third of GHG emissions. However, improved agricultural practices can help mitigate climate change by reducing emissions from agriculture and other sources and by storing carbon in soils.

The development of agriculture during the past centuries and particularly in last decades has entailed depletion of substantive soil carbon stocks. Agricultural soils are among the planet's largest reservoirs of carbon and hold potential for expanded carbon sequestration (CS), and thus provide a prospective way of mitigating the increasing atmospheric concentration of CO2. There is general agreement that the technical potential for sequestration of carbon in soil is significant, and some consensus on the magnitude of that potential. Croplands worldwide could sequester between 0.90 and 1.85 Pg C/yr, i.e. 26-53% of the target of the “4p1000 Initiative: Soils for Food Security and Climate”.

At the same time, this process provides other important benefits for soil, crop and environment quality, prevention of erosion and desertification, and for the enhancement of biodiversity. Land degradation does not only reduce crop yields but often reduces the carbon content of agro ecosystems, and may reduce biodiversity.

The climate of earth has been experiencing an unprecedented change due to the rapidly increasing amount of GHGs in the atmosphere. There is a need to devise multiple strategies to offset the current release of GHGs into atmosphere. CO ₂ has a prominent share in global warming amongst all GHGs in atmosphere. Soil carbon sequestration is a promising approach to offset the rising amount of CO ₂ in the atmosphere. Both partially degraded and agricultural soils have a considerable potential to minimise the elevated CO ₂ levels in the atmosphere. On a global scale, the soils can retain two-fold more carbon than that present in the atmosphere or captured in vegetation. The temperature, soil moisture and elevated CO ₂ levels are the dominant climatic factors affecting the soil carbon sequestration. Soil carbon sequestration is also strongly influenced by various edaphic factors i.e. soil texture, soil structure, soil porosity, soil compaction, soil mineralogy, and soil microbial community composition etc. Additionally, agricultural practices like land-use changes, plant residue management, agro-chemicals etc. influence soil organic carbon (SOC) stocks, either directly (e.g. by altering the amount of carbon being added in the soil) or indirectly (e.g. by influencing soil aggregation and thereby accelerating microbial decomposition processes). Besides offsetting the rapidly increasing atmospheric GHGs, soil carbon sequestration may potentially improve the soil quality and advances the food security. It may play a crucial role in sustainable agriculture because it is highly sustainable and an environmentally friendly approach. It can enhance the soil quality by improving soil health parameters (i.e. water retaining capacity of soil) followed by improved crop production on sustainable basis.

The present invention has been devised in light of the above considerations.

Summary of the Invention

The invention is based on overexpression of protein components responsible for the synthesis and secretion of cellulose in microorganisms such as root-associated bacteria to achieve an increase in water retention around plant roots. This increase in water retention around plant roots is thought to reduce the amount of water irrigation required, and therefore improve the crop’s tolerance to environmental drought.

Thus the invention at its broadest provides a genetically engineered microorganism modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose relative to a reference microorganism, optionally wherein the cellulose is bacterial cellulose. Preferably, the microorganism (and reference microorganism) is a bacterium such as a root-associated bacterium. In one aspect of the invention, provided is a genetically engineered microorganism for producing cellulose, wherein the microorganism is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose, wherein the microorganism is modified with exogenous genes comprising a bcsA gene, a bcsB gene, a bcsC gene, a bcsD gene, and a ccpAx gene. In some embodiments, the microorganism is further modified with an exogenous cmcAx gene and/or an exogenous bglAx gene. In some embodiments, the microorganism is modified with an exogenous nucleic acid comprising a bcsA gene, a bcsB gene, a bcsC gene, a bcsD gene, and at least a ccpAx gene. In some embodiments, the genes are heterologous. In some embodiments, the genes are each isolated from K. xylinus.

In some embodiments, the genetically engineered microorganism is a bacterium, optionally a root-associated bacterium. In some embodiments, the genetically engineered microorganism is a plant growth-promoting rhizobacterium. In some embodiments, the microorganism is a Pseudomonas bacterium. In some embodiments, the rhizobacterium is not Komagataeibacter xylinus (also known as Acetobacter xylinum and Gluconacetobacter xylinus). In some embodiments, expression of the genes is regulated by a cell-density quorum sensing system.

In some embodiments, a quorum sensing operon is instered into the host cell. In some embodiments, the quorum sensing system comprises a gene encoding a sensor kinase and a gene encoding a response regulator. In further embodiments, the quorum sensing system further comprises a quorum sensing regulated promoter. In alternative embodiments, the quorum sensing system comprises a gene encoding a signalling molecule (autoinducer) and a gene encoding a transcriptional/response regulator. In further embodiments, the quorum sensing system further comprises a quorum sensing regulated promoter.

In some aspects, provided is a method of increasing production of cellulose in a microorganism compared to a reference microorganism, wherein the method comprises a step of modifying the microorganism to overexpress at least one protein involved in synthesis and/or secretion of cellulose, wherein the microorganism is modified with exogenous genes comprising a bcsA gene, a bcsB gene, a bcsC gene, a bcsD gene, and a ccp gene. In some embodiments, the microorganism is further modified with an exogenous cmc gene and/or an exogenous bgl gene. In some embodiments, the microorganism is a bacterium, optionally a plant growth-promoting rhizobacterium.

The invention provides a genetically engineered microorganism for producing cellulose, wherein the microorganism is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose. In some embodiments, the cellulose produced by the genetically engineered microorganism is bacterial cellulose. In some embodiments, the microorganism is genetically modified to overexpress at least one protein from a cellulose synthase complex. In some embodiments, the microorganism is modified to overexpress at least one, at least two, at least three, or at least four of the proteins from a cellulose synthase complex. In some embodiments, the cellulose synthase complex is a bacterial cellulose synthase complex. In some embodiments, cellulose production is increased in the genetically modified microorganism compared to a reference microorganism. In some embodiments, the reference microorganism is of the same species as the modified microorganism. The reference microorganism may be the same strain as the modified microorganism. In some embodiments, the microorganism is a wild-type microorganism. In some embodiments, the reference microorganism of the same species or same strain is a wild-type microorganism of the same species or same strain. In some embodiments, the genetically engineered microorganism is selected from a bacterial cell, a fungal cell or an algae cell. In some embodiments, the genetically engineered microorganism is a bacterium. In further embodiments, the genetically engineered microorganism is a root-associated bacterium.

In some embodiments, the genetically engineered microorganism of the invention is modified to overexpress a cellulose synthase complex. In some embodiments, the genetically modified microorganism is modified with an exogenous nucleic acid encoding at least one protein from a cellulose synthase complex. In other embodiments, overexpression of at least one protein of a cellulose synthase complex is achieved by increasing transcription and/or translation of the at least one protein of an endogenous cellulose synthase complex.

In some embodiments, the genetically modified microorganism is modified with an exogenous nucleic acid encoding at least one protein from a cellulose synthase complex. In some embodiments, the microorganism is modified to with an exogenous nucleic acid encoding at least one, at least two, at least three, or at least four of the proteins from a cellulose synthase complex. In some embodiments, the genetically engineered microorganism is modified with at least one of the following genes of the bcs operon: bcsA ; bcsB; bcsC; and/or bcsD. In further embodiments, the exogenous nucleic acid comprises a bcs operon. In another further embodiment, the bcs operon encodes four protein subunits BcsA, BcsB, BcsC, and BcsD. In some embodiments, the exogenous nucleic acid further comprises at least one of the following genes or operon: cmcAx gene; ccpAx gene; bglAx gene; pgm gene; galU gene; cdg operon; and/or dgc gene. In some embodiments, the exogenous nucleic acid comprises a bcs operon, a cmcAx gene, a ccpAx gene, and a bglAx gene. In some embodiments, the exogenous nucleic acid comprises a bcs operon, a cmc gene, a ccp gene, a bgl gene, a pgm gene, a galU gene, a cdg operon and a dgc gene. In some embodiments, the exogenous nucleic acid consists of a bcs operon, a cmc gene, a ccp gene, a bgl gene, a pgm gene, a galU gene, a cdg operon and a dgc gene. In some embodiments, the bcs operon, cmc gene, ccp gene, bgl gene, pgm gene, galU gene, cdg operon, and/or dgc gene are each isolated from K. xylinus.

In some embodiments, the microorganism is selected from Pseudomonas fluorescens, and Bacillus megaterium. In a further embodiment, the microorganism is Pseudomonas fluorescens. In another further embodiment, the microorganism is Pseudomonas fluorescens SBW25. In another further embodiment, the microorganism is Pseudomonas fluorescens F113. In another further embodiment, the microorganism is Pseudomonas fluorescens CHAO. In another further embodiment, the microorganism is Pseudomonas fluorescens Pf-5. In another further embodiment, the microorganism is Pseudomonas fluorescens FW300 N2E2.

In some embodiments, the cellulose produced by the genetically engineered microorganism of the invention is secreted outside of the cell. In a further embodiment, the secreted cellulose forms a network outside of the cell. In some embodiments, the secreted network forms around plant roots. In some embodiments, the secreted cellulose network increases water retention around plant roots. In some embodiments, the plant is a cereal plant, a corn plant, a rice plant, a wheat plant, or a soy plant.

In a second aspect of the invention, a method of increasing production of cellulose in a microorganism compared to a reference microorganism, wherein the method comprises a step of modifying the microorganism to overexpress at least one protein involved in synthesis and/or secretion of cellulose. In some embodiments, the microorganism is modified to overexpress at least one protein from a cellulose synthase complex. In some embodiments, the reference microorganism is of the same species as the modified microorganism. In some embodiments, the reference microorganism is a wild-type microorganism. In some embodiments, the reference microorganism of the same species is a wild-type microorganism of the same species. In some embodiments, the genetically engineered microorganism is modified with an exogenous nucleic acid encoding at least one protein from a cellulose synthase complex. In some embodiments, the exogenous nucleic acid encoding at least one protein from a cellulose synthase complex is integrated into the genome of the microorganism. In some embodiments, the cellulose is bacterial cellulose. In some embodiments the exogenous nucleic acid comprises a bcs operon. In further embodiments, the exogenous nucleic acid of the vector further comprises at least one of cmcAx gene, ccpAx gene, bglAx gene, pgm gene, a galU gene, a cdg operon, and a dgc gene. In some embodiments, the genetically engineered microorganism is selected from a bacterial cell, a fungal cell or an algae cell. In some embodiments, the genetically engineered microorganism is a bacterium. In further embodiments, the genetically engineered microorganism is a root-associated bacterium. In a third aspect of the invention, a vector comprising an exogenous nucleic acid that encodes at least one protein from a cellulose synthase complex is provided. In some embodiments, the exogenous nucleic acid of the vector comprises a bcs operon. In further embodiments, the exogenous nucleic acid of the vector further comprises at least one of a cmcAx gene, a ccpAx gene, a bglAx gene, a pgm gene, a galU gene, a cdg operon, and a dgc gene. In some embodiments, the exogenous nucleic acid of the vector comprises a bcs operon, a cmcAx gene, a ccpAx gene, and a bglAx gene. In some embodiments, the exogenous nucleic acid of the vector comprises a bcs operon, a cmcAx gene, a ccpAx gene, a bglAx gene, a pgm gene, a galU gene, a cdg operon and a dgc gene. In some embodiments, the exogenous nucleic acid of the vector consists of a bcs operon, a cmcAx gene, a ccpAx gene, a bglAx gene, a pgm gene, a galU gene, a cdg operon and a dgc gene. In some embodiments, the bcs operon, cmcAx gene, ccpAx gene, bglAx gene, pgm gene, galU gene, cdg operon, and/or dgc gene are each isolated from K. xylinus. In some embodiments, the vector is an isolated vector. In some embodiments, the genes are heterologous.

In a fourth aspect, the invention provides a method of producing a genetically engineered microorganism for producing cellulose, wherein the method comprises a step of modifying the microorganism with an exogenous nucleic acid that encodes at least one protein from a cellulose synthase complex comprising: a) isolating a microorganism; and b) introducing the vector of the invention into the microorganism.

In some embodiments, the microorganism is modified with an exogenous nucleic acid encoding at least one, at least two, at least three, or at least four of the proteins from a cellulose synthase complex. In some embodiments, the genetically engineered microorganism is selected from a bacterial cell, a fungal cell or an algae cell. In some embodiments, the genetically engineered microorganism is a bacterium. In further embodiments, the genetically engineered microorganism is a root-associated bacterium.

In some embodiments, the vector of the invention is introduced into the microorganism by electroporation. In some embodiments, the vector of the invention is introduced into the microorganism by transfection. In some embodiments, the exogenous nucleic acid encoding at least one protein from a cellulose synthase complex is integrated into the genome of the microorganism. In some embodiments, at least one, at least two, at least three, or at least four of the proteins from a cellulose synthase complex is integrated into the genome of the microorganism. In some embodiments, the vector of the invention is introduced into the microorganism such that two copies, three copies, or four copies and so on are integrated into the genome of the microorganism to increase the copy number of that gene or genes. In some embodiments, cellulose production is increased in the genetically modified microorganism compared to a reference microorganism. In some embodiments, the reference microorganism is of the same species or strain. In some embodiments, the reference microorganism is a wild- type microorganism. In some embodiments, the reference microorganism is a wild-type microorganism of the same species or strain. In some embodiments, the cellulose is bacterial cellulose. In some embodiments, the cellulose synthase complex is a bacterial cellulose synthase complex.

In a fifth aspect, provided is a genetically engineered microorganism obtainable by the method of producing a genetically engineered microorganism for producing cellulose. In another aspect, the invention provides an isolated genetically engineered microorganism of the invention. In an alternative aspect of the invention, provided is a population comprising the genetically engineered microorganism of the invention. In some embodiments, the genetically engineered microorganism is selected from a bacterial cell, a fungal cell or an algae cell. In some embodiments, the genetically engineered microorganism is a bacterium. In further embodiments, the genetically engineered microorganism is a root-associated bacterium.

In another aspect, the invention provides a composition comprising the genetically engineered population of microorganisms of the invention. In some embodiments, the composition is applied to a plant in a liquid formulation. In alternative embodiments, the composition is applied to a plant as an inoculum. In some embodiments, the inoculants are peat-based formulations.

In further embodiments, the formulations are used to coat seeds or pellets for sowing in furrows. In some embodiments, the genetically modified microorganism of the invention is delivered to plants in microbeads. In further embodiments, the microbeads are alginate microbeads. In some embodiments, the composition further comprises a fertiliser and/or a biofertiliser. In some embodiments, the composition is applied to a plant after planting but before harvest of said plant. In some embodiments, the composition is applied to the soil before planting a plant. In some embodiments, the plant is a cereal plant, a corn plant, a rice plant, a wheat plant, or a soy plant. In some embodiments, the genetically engineered microorganism is selected from a bacterial cell, a fungal cell or an algae cell. In some embodiments, the genetically engineered microorganism is a bacterium. In further embodiments, the genetically engineered microorganism is a root-associated bacterium.

In another aspect, the invention provides a method of increasing water-retention around plant roots, comprising applying a genetically engineered microorganism to soil surrounding a plant root, wherein the microorganism is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose. In some embodiments, the microorganism is selected from the genetically engineered microorganism of the invention, the isolated genetically engineered microorganism of the invention, the population of genetically engineered microorganisms of the invention, or the composition of the invention. In some embodiments, the plant is a cereal plant, a corn plant, a rice plant, a wheat plant, or a soy plant. In some embodiments, the genetically engineered microorganism is selected from a bacterial cell, a fungal cell or an algae cell. In some embodiments, the genetically engineered microorganism is a bacterium. In further embodiments, the genetically engineered microorganism is a root- associated bacterium.

In another aspect, the invention provides a method of reducing water consumption in agriculture, comprising applying a genetically engineered microorganism to soil surrounding a plant root, wherein the microorganism is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose. In some embodiments, the microorganism is selected from the genetically engineered microorganism of the invention, the isolated genetically engineered microorganism of the invention, the population of genetically engineered microorganisms of the invention, or the composition of the invention. In some embodiments, the plant is a cereal plant, a corn plant, a rice plant, a wheat plant, or a soy plant. In some embodiments, the genetically engineered microorganism is selected from a bacterial cell, a fungal cell or an algae cell. In some embodiments, the genetically engineered microorganism is a bacterium. In further embodiments, the genetically engineered microorganism is a root- associated bacterium.

In another aspect, the invention provides a plant comprising the genetically engineered microorganism of the invention, the isolated genetically engineered microorganism of the invention, the population of genetically engineered microorganisms of the invention, or the composition of the invention, wherein the genetically engineered microorganism, isolated genetically engineered microorganism, or the population of genetically engineered microorganisms is associated with the plant roots. In some embodiments, the plant is a cereal plant, a corn plant, a rice plant, a wheat plant, or a soy plant. In some embodiments, the genetically engineered microorganism is selected from a bacterial cell, a fungal cell or an algae cell. In some embodiments, the genetically engineered microorganism is a bacterium. In further embodiments, the genetically engineered microorganism is a root-associated bacterium.

In another aspect, the invention provides a method of capturing carbon, comprising applying a genetically engineered microorganism to soil surrounding a plant root, wherein the microorganism is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose, and wherein the carbon is converted to cellulose by the microorganism.

In some embodiments the genetically engineered microorganism is a microorganism of the invention, an isolated genetically engineered microorganism of the invention, a population of genetically engineered microorganisms of the invention, or a composition of the invention. In some embodiments, the carbon is absorbed as carbohydrates secreted from a plant and the carbohydrates are converted into cellulose by the microorganism. In some embodiments, production of cellulose results in increased water-retention around plant roots. In some embodiments, the genetically engineered microorganism is selected from a bacterial cell, a fungal cell or an algae cell. In some embodiments the genetically engineered microorganism is a bacterium. In further embodiments, the genetically engineered microorganism is a root- associated bacterium. In some embodiments, the microorganism is a mycorrhizal fungi (for example, arbuscular, ectomycorrhizal, ericoid and/or orchid).

Another aspect of the invention provides use of a genetically modified microorganism in agriculture, wherein the microorganism is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose. In another aspect of the invention, use of a genetically modified microorganism to increase water-retention around plant roots, wherein the microorganism is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose, is provided. Another aspect of the invention provides use of a genetically modified microorganism in carbon capture, wherein the microorganism is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose, and wherein the carbon is converted into cellulose by the microorganism. In some embodiments, the genetically engineered microorganism is selected from a bacterial cell, a fungal cell or an algae cell. In some embodiments, the genetically engineered microorganism is a bacterium. In further embodiments, the genetically engineered microorganism is a root- associated bacterium. In some embodiments, the methods and uses described herein result in an increase in plant viability.

In some embodiments, provided is a genetically modified microorganism comprising one or more heterologous genes, wherein the genes comprise a bcsA gene, a bcsB gene, a bcsC gene, a bcsD gene, a cmcAx gene, a ccpAx gene, and/or a bglAx gene. In some embodiments, the microorganism is a bacterium. In further embodiments, the microorganism is a plant growth promoting rhizobacterium.

In some aspects, the invention provides a genetically engineered root-associated bacterium for producing cellulose, wherein the bacterium is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose. In some embodiments, the cellulose produced by the genetically engineered root-associated bacterium is bacterial cellulose. In some embodiments, the bacterium is genetically modified to overexpress at least one protein from a cellulose synthase complex. In some embodiments, the bacteria is modified to overexpress at least one, at least two, at least three, or at least four of the proteins from a cellulose synthase complex. In some embodiments, the cellulose synthase complex is a bacterial cellulose synthase complex. In some embodiments, cellulose production is increased in the genetically modified bacterium compared to a reference bacterium. In some embodiments, the reference bacterium is of the same species as the modified bacterium. The reference bacterium may be the same strain as the modified bacterium. In some embodiments, the bacterium is a wild-type bacterium. In some embodiments, the reference bacterium of the same species or same strain is a wild-type bacterium of the same species or same strain.

In some embodiments, the genetically engineered root-associated bacterium of the invention is modified to overexpress a cellulose synthase complex. In some embodiments, the genetically modified bacterium is modified with an exogenous nucleic acid encoding at least one protein from a cellulose synthase complex. In other embodiments, overexpression of at least one protein of a cellulose synthase complex is achieved by increasing transcription and/or translation of the at least one protein of an endogenous cellulose synthase complex.

In some embodiments, the genetically modified root-associated bacterium is modified with an exogenous nucleic acid encoding at least one protein from a cellulose synthase complex. In some embodiments, the bacteria is modified to with an exogenous nucleic acid encoding at least one, at least two, at least three, or at least four of the proteins from a cellulose synthase complex. In some embodiments, the genetically engineered bacterium is modified with at least one of the following genes of the bcs operon: bcsA ; bcsB; bcsC; and/or bcsD. In further embodiments, the exogenous nucleic acid comprises a bcs operon. In another further embodiment, the bcs operon encodes four protein subunits BcsA, BcsB, BcsC, and BcsD. In some embodiments, the exogenous nucleic acid further comprises at least one of the following genes or operon: cmcAx gene; ccpAx gene; bglAx gene; pgm gene; galU gene; cdg operon; and/or dgc gene. In some embodiments, the exogenous nucleic acid comprises a bcs operon, a cmcAx gene, a ccpAx gene, and a bglAx gene. In some embodiments, the exogenous nucleic acid comprises a bcs operon, a cmcAx gene, a ccpAx gene, a bglAx gene, a pgm gene, a galU gene, a cdg operon and a dgc gene. In some embodiments, the exogenous nucleic acid consists of a bcs operon, a cmcAx gene, a ccpAx gene, a bglAx gene, a pgm gene, a galU gene, a cdg operon and a dgc gene. In some embodiments, the bcs operon, cmcAx gene, ccpAx gene, bglAx gene, pgm gene, galU gene, cdg operon, and/or dgc gene are each isolated from K. xylinus.

In some embodiments, the root-associated bacterium is selected from Pseudomonas fluorescens, and Bacillus megaterium. In a further embodiment, the root-associated bacterium is Pseudomonas fluorescens. In another further embodiment, the root-associated bacterium is Pseudomonas fluorescens SBW25. In another further embodiment, the root-associated bacterium is Pseudomonas fluorescens F113. In another further embodiment, the root- associated bacterium is Pseudomonas fluorescens CHAO. In another further embodiment, the root-associated bacterium is Pseudomonas fluorescens Pf-5. In another further embodiment, the root-associated bacterium is Pseudomonas fluorescens FW300 N2E2.

In some embodiments, the cellulose produced by the genetically engineered root-associated bacterium of the invention is secreted outside of the cell. In a further embodiment, the secreted cellulose forms a network outside of the cell. In some embodiments, the secreted network forms around plant roots. In some embodiments, the secreted cellulose network increases water retention around plant roots. In some embodiments, the plant is a cereal plant, a corn plant, a rice plant, a wheat plant, or a soy plant.

In a second aspect of the invention, a method of increasing production of cellulose in a root- associated bacterium compared to a reference root-associated bacterium, wherein the method comprises a step of modifying the bacterium to overexpress at least one protein involved in synthesis and/or secretion of cellulose. In some embodiments, the bacterium is modified to overexpress at least one protein from a cellulose synthase complex. In some embodiments, the reference bacterium is of the same species as the modified bacterium. In some embodiments, the reference bacterium is a wild-type bacterium. In some embodiments, the reference bacterium of the same species is a wild-type bacterium of the same species. In some embodiments, the genetically engineered root-associated bacterium is modified with an exogenous nucleic acid encoding at least one protein from a cellulose synthase complex. In some embodiments, the exogenous nucleic acid encoding at least one protein from a cellulose synthase complex is integrated into the genome of the root-associated bacterium. In some embodiments, the cellulose is bacterial cellulose. In some embodiments the exogenous nucleic acid comprises a bcs operon. In further embodiments, the exogenous nucleic acid of the vector further comprises at least one of cmcAx gene, ccpAx gene, bglAx gene, pgm gene, a galU gene, a cdg operon, and a dgc gene.

In a third aspect of the invention, a vector comprising an exogenous nucleic acid that encodes at least one protein from a cellulose synthase complex is provided. In some embodiments, the exogenous nucleic acid of the vector comprises a bcs operon. In further embodiments, the exogenous nucleic acid of the vector further comprises at least one of a cmcAx gene, a ccpAx gene, a bglAx gene, a pgm gene, a galU gene, a cdg operon, and a dgc gene. In some embodiments, the exogenous nucleic acid of the vector comprises a bcs operon, a cmcAx gene, a ccpAx gene, and a bglAx gene. In some embodiments, the exogenous nucleic acid of the vector comprises a bcs operon, a cmcAx gene, a ccpAx gene, a bglAx gene, a pgm gene, a galU gene, a cdg operon and a dgc gene. In some embodiments, the exogenous nucleic acid of the vector consists of a bcs operon, a cmcAx gene, a ccpAx gene, a bglAx gene, a pgm gene, a galU gene, a cdg operon and a dgc gene. In some embodiments, the bcs operon, cmcAx gene, ccpAx gene, bglAx gene, pgm gene, galU gene, cdg operon, and/or dgc gene are each isolated from K. xylinus. In some embodiments, the vector is an isolated vector.

In a fourth aspect, the invention provides a method of producing a genetically engineered root- associated bacterium for producing cellulose, wherein the method comprises a step of modifying the bacterium with an exogenous nucleic acid that encodes at least one protein from a cellulose synthase complex comprising: a) isolating a root-associated bacterium; and b) introducing the vector of the invention into the root-associated bacterium.

In some embodiments, the bacteria is modified with an exogenous nucleic acid encoding at least one, at least two, at least three, or at least four of the proteins from a cellulose synthase complex.

In some embodiments, the vector of the invention is introduced into the root-associated bacterium by electroporation. In some embodiments, the vector of the invention is introduced into the root-associated bacterium by transfection. In some embodiments, the exogenous nucleic acid encoding at least one protein from a cellulose synthase complex is integrated into the genome of the root-associated bacterium. In some embodiments, at least one, at least two, at least three, or at least four of the proteins from a cellulose synthase complex is integrated into the genome of the root-associated bacterium. In some embodiments, the vector of the invention is introduced into the bacterium such that two copies, three copies, or four copies and so on are integrated into the genome of the bacterium to increase the copy number of that gene or genes. In some embodiments, cellulose production is increased in the genetically modified bacterium compared to a reference bacterium. In some embodiments, the reference bacterium is of the same species or strain. In some embodiments, the reference bacterium is a wild-type bacterium. In some embodiments, the reference bacterium is a wild-type bacterium of the same species or strain. In some embodiments, the cellulose is bacterial cellulose. In some embodiments, the cellulose synthase complex is a bacterial cellulose synthase complex.

In a fifth aspect, provided is a genetically engineered root-associated bacterium obtainable by the method of producing a genetically engineered root-associated bacterium for producing cellulose. In another aspect, the invention provides an isolated genetically engineered root- associated bacterium of the invention. In an alternative aspect of the invention, provided is a bacterial population comprising the genetically engineered root-associated bacterium of the invention.

In another aspect, the invention provides a bacterial composition comprising the genetically engineered root-associated bacterial population of the invention. In some embodiments, the composition is applied to a plant in a liquid formulation. In alternative embodiments, the composition is applied to a plant as a bacterial inoculum. In some embodiments, the bacterial inoculants are peat-based formulations. In further embodiments, the formulations are used to coat seeds or pellets for sowing in furrows. In some embodiments, the genetically modified bacterium of the invention is delivered to plants in microbeads. In further embodiments, the microbeads are alginate microbeads. In some embodiments, the bacterial composition further comprises a fertiliser and/or a biofertiliser. In some embodiments, the bacterial composition is applied to a plant after planting but before harvest of said plant. In some embodiments, the bacterial composition is applied to the soil before planting a plant. In some embodiments, the plant is a cereal plant, a corn plant, a rice plant, a wheat plant, or a soy plant.

In another aspect, the invention provides a method of increasing water-retention around plant roots, comprising applying the genetically engineered root-associated bacteria of the invention, the isolated genetically engineered root-associated bacterium of the invention, the population of genetically engineered root-associated bacteria of the invention, or the bacterial composition of the invention to the surrounding soil of the plant. In some embodiments, the plant is a cereal plant, a corn plant, a rice plant, a wheat plant, or a soy plant.

In another aspect, the invention provides a method of reducing water consumption in agriculture, comprising applying the genetically engineered root-associated bacteria of the invention, the isolated genetically engineered root-associated bacterium of the invention, the population of genetically engineered root-associated bacteria of the invention, or the bacterial composition of the invention to the surrounding soil of the plant. In some embodiments, the plant is a cereal plant, a corn plant, a rice plant, a wheat plant, or a soy plant.

In another aspect, the invention provides a plant comprising the genetically engineered root- associated bacteria of the invention, the isolated genetically engineered root-associated bacterium of the invention, the population of genetically engineered root-associated bacteria of the invention, or the bacterial composition of the invention, wherein the genetically engineered root-associated bacterium, isolated genetically engineered root-associated bacterium, or the population of genetically engineered root-associated bacteria is associated with the plant roots. In some embodiments, the plant is a cereal plant, a corn plant, a rice plant, a wheat plant, or a soy plant.

In another aspect, the invention provides a method of capturing carbon, comprising applying a genetically engineered root-associated bacterium to soil surrounding a plant root, wherein the bacterium is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose, and wherein the carbon is converted to cellulose by the bacterium. In some embodiments the genetically engineered root-associated bacterium is a root- associated bacterium of the invention, an isolated genetically engineered root-associated bacterium of the invention, a population of genetically engineered root-associated bacteria of the invention, or a bacterial composition of the invention. In some embodiments, the carbon is absorbed as carbohydrates secreted from a plant and the carbohydrates are converted into cellulose by the bacterium. In some embodiments, production of cellulose results in increased water-retention around plant roots.

Another aspect of the invention provides use of a genetically modified root-associated bacterium in agriculture, wherein the bacterium is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose. In another aspect of the invention, use of a genetically modified root-associated bacterium to increase water-retention around plant roots, wherein the bacterium is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose, is provided. Another aspect of the invention provides use of a genetically modified root-associated bacterium in carbon capture, wherein the bacterium is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose, and wherein the carbon is converted into cellulose by the bacterium. In some embodiments, the methods and uses described herein result in an increase in plant viability.

In some embodiments, the methods described herein result in an increase in plant viability.

In some embodiments of the invention, the root-associated bacterium is genetically modified with an exogenous nucleic acid comprising one or more heterologous genes, wherein the genes are selected from: a bcsA gene, a bcsB gene, a bcsC gene, a bcsD gene, a cmcAx gene, a ccpAx gene, and a bglAx gene.

In some aspects, the invention provides a genetically modified bacterium for producing cellulose, wherein the bacterium is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose, and wherein the genetically modified bacterium is modified with an exogenous bcs operon, wherein the bcs operon comprises a bcsA gene, a bcsB gene, a bcsC gene, and a bcsD gene. In some embodiments, provided is a genetically engineered bacterium, wherein the bacterium is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose, and wherein the genetically modified bacterium is modified with one or more heterologous genes, wherein the genes comprise a bcsA gene, a bcsB gene, a bcsC gene, a bcsD gene, and optionally a cmcAx gene, a ccpAx gene, and/or a bglAx gene. In some embodiments, the bacterium is not Komagataeibacter xylinus (also known as Acetobacter xylinum and Gluconacetobacter xylinus). In some embodiments of the invention, the bacterium is genetically modified with an exogenous nucleic acid comprising one or more heterologous genes, wherein the genes are selected from: a bcsA gene, a bcsB gene, a bcsC gene, a bcsD gene, a cmcAx gene, a ccpAx gene, and a bglAx gene.

In some aspects, the invention provides a genetically engineered plant growth- promoting rhizobacterium for producing cellulose, wherein the rhizobacterium is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose. In some embodiments, the rhizobacterium is modified with an exogenous nucleic acid comprising a bcs operon, wherein the bcs operon comprises a bcsA gene, a bcsB gene, a bcsC gene, and a bcsD gene. In some embodiments, the exogenous nucleic acid further comprises at least a ccpAx gene. In some embodiments, the exogenous nucleic acid further comprises a cmcAx gene, a ccpAx gene, and/or a bglAx gene. In some embodiments, the genes are heterologous. In some embodiments of the invention, the rhizobacterium is genetically modified with an exogenous nucleic acid comprising one or more heterologous genes, wherein the genes are selected from: a bcsA gene, a bcsB gene, a bcsC gene, a bcsD gene, a cmcAx gene, a ccpAx gene, and a bglAx gene.

In some embodiments, provided is a genetically engineered plant growth-promoting rhizobacterium for producing cellulose, wherein the rhizobacterium is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose, and wherein the genetically modified rhizobacterium is modified with one or more heterologous genes, wherein the genes comprise a bcsA gene, a bcsB gene, a bcsC gene, a bcsD gene, and optionally a cmcAx gene, a ccpAx gene, and/or a bglAx gene. In some embodiments, the rhizobacterium is not Komagataeibacter xylinus (also known as Acetobacter xylinum and Gluconacetobacter xylinus) .

The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

Summary of the Figures

Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

Figure 1. A depiction of the genetic crossover from the shuttle vector (pEX18Ap) containing the bacterial cellulose genes of interest onto the chromosome of P. fluorescens, such as P. fluorescens SBW25, at locus -6- Figure 2. A depiction of the genetic crossover from the Mini CTX1 vector containing the bacterial cellulose genes of interest onto the chromosome of P. fluorescens at the attb site (attB

- 5’ TGAGTTCGAATCTCACCGCCTCCGCCATAT 3’).

Figure 3. A depiction of a construct comprising the cellulose synthesis genes cmcAx, ccpAx, BcsA, BcsB, BcsC, BcsD and BglAx, and GFP as a reporter gene. In this particular example, the construct also comprises a pBAD promoter and a pBAD terminator.

Figure 4. a) A depiction of a construction comprising Pseudomonas synxantha strain 2-79 chromosome - the phzl/R operon. b) A depiction of the construct comprising for insertion into the host microorganism, for example Pseudomonas fluorescens.

Figure 5. a) A depiction of a construct comprising Pseudomonas putida strain KT2440 chromosome rox quorum sensing system b) A depiction of a construct comprising the KT2440 QS-system for recombinant protein production in Pseudomonas, utilizing the regions upstream of the roxS/roxR-regulated genes shown in Figure 5c. c) A depiction of a construct for insertion into the host microorganism, for example Pseudomonas fluorescens.

Detailed Description of the Invention

Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

The present invention provides genetically engineered microorganisms such as a root- associated bacterium for producing cellulose, wherein the genetically engineered root- associated bacterium is genetically modified to increase cellulose production relative to a reference bacterium, typically of the same species. Typically, the genetically engineered root- associated bacterium are modified to overexpress proteins required for synthesis and/or secretion of cellulose, preferably a cellulose synthase complex.

As used herein “root-associated” bacterium or bacteria refers to a bacterium/bacteria that live on the plant root or surrounding the plant root. In some embodiments, the microorganisms are “root-associated”, referring to a microorganism that lives on the plant root or surrounding the plant root. In further embodiments, the root-associated bacteria are Rhizobacteria. The root- associated bacteria may form symbiotic relationships with plants, promoting plant growth.

(Such plant growth-promoting rhizobacteria are termed PGPR.) Without being bound by theory it is anticipated that once the root-associated bacteria are detached from the root, the bacterium would be unable to sustain viability, and so are unlikely to survive in the wider environment thereby preventing the spread of the genetically modified bacteria in the environment. Examples of root-associated bacteria include, but are not limited to, Agrobacterhim radiohacter, Bacillus acidocaldarms, Bacillus acidoterresiris, Bacillus agri, Bacillus aizawai, Bacillus albolactis, Bacillus alcalophilus, Bacillus alvei, Bacillus aminoglucosidicus, Bacillus aminovorans, Bacillus amylolyticus (also known as Paenibacilhis amylolyticus ), Bacillus amyloliquefacieris, Bacillus aneiirinolyticus, Bacillus atropkaeus, Bacillus azotoformans, Bacillus badius, Bacillus cereus (synonyms: Bacillus endorhythmos, Bacillus medusa), Bacillus chiiinosporus, Bacillus circulans, Bacillus coagulans, Bacillus endoparasiticus, Bacillus fastidiosus, Bacillus firmus, Bacillus kurstaki, Bacilhis lacticola, Bacilhis laclimorbus, Bacillus laciis, Bacillus laierospoms (also known as Brevibacillus laterosporus), Bacillus lautus, Bacillus leniimorbus, Bacillus lenius, Bacillus licheniformis, Bacillus maroccanus, Bacillus megaterium, Bacillus meiiens, Bacillus mycoides, Bacillus natto, Bacillus nematocida, Bacillus nigrificans, Bacillus nigrum, Bacillus pantothenticus, Bacillus papillae, Bacillus psychrosaccharolyticus, Bacillus pumilus, Bacillus siamensis, Bacillus smithii, Bacillus sphaericus, Bacillus subiilis, Bacillus thuringiensis, Bacillus tmiflagellatus, Bradyrhizobium japonicum, Brevibacillus brevis, Brevibacillus laterosporus (formerly Bacillus laterosporus), Chromobacterium suhisugae, Delflia acidovorans, Lactobacillus acidophilus, Lysobacter antibioticus, Lysobacter enzymogenes, Paenibacilhis alvei, Paenibacillus polymyxa, Paenibacillus popilliae (formerly Bacillus popilliae ), Pantoea agglomerans, Pasteuria penetrans (formerly Bacillus penetrans), Pasteuria usgae, Pecto bacterium carotovorum (formerly Erwinia carotovord), Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas cepacia (formerly known as Burkholderia cepacia), Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas proradix, Pseudomonas putida, Pseudomonas syringae, Serraiia entomophila, Serratia marcescens, Streptomyces colombiensis, Streptomyces galbus, Streptomyces goshikiensis, Streptomyces griseoviridis, Streptomyces lavendulae, Streptomyces prasinus, Streptomyces saraceticus, Streptomyces venezuelae, Xanthomonas campestris, Xenorhabdus luminescens, Xenorhabdus nematophila, Rhodococcus globerulus AQ719 (NRRL Accession No. B-21663), Bacillus sp. AQ175 (ATCC Accession No. 55608), Bacillus sp. AQ 177 (ATCC Accession No. 55609), Bacillus sp. AQ178 (ATCC Accession No. 53522), and Streptomyces sp. strain NRRL Accession No. B-30145.

In some embodiments, the bacterium Pseudomonas fluorescens or Bacillus megaterium. In a further embodiment, the bacterium is Pseudomonas fluorescens. In preferred embodiments, the bacterium is Pseudomonas fluorescens SWB25. In another further embodiment, the bacterium is Pseudomonas fluorescens F113. In another further embodiment, the bacterium is Pseudomonas fluorescens CHAO. In another further embodiment, the bacterium is Pseudomonas fluorescens Pf-5. In another further embodiment, the bacterium is Pseudomonas fluorescens FW300 N2E2.

Rhizobacteria colonise the surface of the root, or superficial intercellular space of the host plant, often forming root nodules. In some embodiments, the root-associated bacteria are plant growth-promoting rhizobacteria (PGPR). Some common examples of PGPR genera exhibiting plant growth promoting activity are: Pseudomonas, Azospirillum, Bacillus, etc. Other known PGPRs include Mesorhizobium ciceri, Burkholderia ambifaria, Mycobacterium phlei, and G. diazotrophicus It is known by the skilled person that PGPR describes soil bacteria that colonise the roots of plants and enhance plant growth. PGPR is not intended to cover bacteria which have a pathogenic effect on the plant, for example, deleterious rhizobacteria (DRB). Six strains of rhizobacteria have been identified as being DRB, these include: the genera Enterobacter, Klebsiella, Citrobacter, Flavobacterium, Achromobacter, and Arthrobacter.

In some embodiments, the bacterium is a gram-negative bacterium. In some embodiments, the bacterium is a Pseudomonas genus bacterium. In some embodiments, the bacterium is not Komagataeibacter xylinus (also known as Acetobacter xylinum and Gluconacetobacter xylinus).

In some embodiments, the bacterium is selected from the following Pseudomonas fluorescens strains: Pseudomonas fluorescens CHAO (CP043179.1); Pseudomonas fluorescens F113 (CP003150.1); Pseudomonas fluorescens FW300 N2E2 (CP015225.1); Pseudomonas fluorescens Pf-275 (CP031648.1); Pseudomonas fluorescens Pf-5 (CP000076.1);

Pseudomonas fluorescens Pf0-1 (CP000094.2); Pseudomonas fluorescens FR1 (CP025738.1); Pseudomonas fluorescens DR133 (CP048607.1); and Pseudomonas fluorescens 2P24 (CP025542.1). Strains CHAO and Pf-5 are now considered to belong to a novel bacterial species Pseudomonas protegens, which are widespread Gram-negative, plant-protecting bacteria. However, in the art these particular strains (CHAO and Pf-5) are also referred to as strains of Pseudomonas fluorescens. Thus, in some instances the bacterium is Pseudomonas protegens, particularly with reference to the strains CHAO and Pf-5. In a further embodiment, the bacterium is a Pseudomonas fluorescens F113.

Cellulose

In some embodiments, the cellulose is bacterial cellulose. In some embodiments, the cellulose produced by the genetically modified microorganism or bacteria is secreted outside of the cell. Without being bound by theory, it is considered that bacterial cellulose has different properties from plant cellulose and is characterised by high purity, strength, moldability and increased water holding ability. It has been demonstrated that plant cellulose has a water retention value of around 60%, while bacterial cellulose has a water retention value of 1000% of the cellulose sample weight (Klemm, et al. 2001). In some embodiments, the secreted bacterial cellulose forms a network around the plant roots. In some embodiments, the bacterial cellulose network forms a spongy network. In some embodiments, the cellulose network is produced around plant roots.

It is anticipated that the network of bacterial cellulose produced by the genetically modified microorganism or bacteria of the invention will facilitate water management. Typically, the bacterial cellulose network retains water and provides an osmotic effect when water is required by the root. Moreover, the bacterial cellulose network may create an environment that increases the soil microbiome, delivering healthier soil and crops. Furthermore, the bacterial cellulose network may have the potential to prevent localised flooding from dramatic weather events. It is further anticipated that the bacterial cellulose network has the ability to act as a bio scaffold to retain a greater quantity of nutrients around the root from the biofertiliser, and prevent run off of the fertiliser.

In some embodiments, the bacterial cellulose retains water. An increase in water retention is anticipated to result in an increase in crop viability and yield. In further embodiments, the bacterial cellulose facilitates a reduction in water evaporation. A reduction in water evaporation will reduce inefficient usage of water. Therefore, it is anticipated that the genetically modified microorganism or bacteria of the invention will increase the amount of water retained around a plant root system and as a result increase crop viability and yield in climates that have reduced rainfall and/or drought. In some embodiments, the genetically engineered microorganism, bacterium or root-associated bacterium is applied to a plant after planting but before harvest of said plant. In some embodiments, the genetically engineered microorganism, bacterium or root- associated bacterium is applied to the soil before planting a plant. In some embodiments, the genetically engineered microorganism, bacterium or root-associated bacterium is applied to the seed of a plant before planting.

The terms “increased cellulose production” and “increasing production of cellulose” are used herein to describe a greater amount of cellulose produced in a genetically engineered microorganism or bacterium compared to a reference microorganism or bacterium, respectively, optionally of the same strain or species. In some embodiments, the reference microorganism is a wild-type microorganism of the same strain or species. In some embodiments, the reference bacterium is a wild-type bacterium of the same strain or species. The increase in cellulose production may be 2-fold, 3-fold, 4-fold, 5-fold, 6-fold and so on, compared to a reference microorganism or bacterium. The quantity of cellulose produced by the genetically engineered microorganism or bacteria can be quantified by techniques that determine the weight of the dried and/or wet cellulose biomass. It is anticipated that the genetically engineered microorganism or bacterium of the invention will produce an increased dried and/or wet weight of cellulose biomass compared to a reference microorganism or bacterium, respectively. Bacterial cellulose may be quantified as described by Jozala A. F., et al. 2014 (which is incorporated by reference). For example, the bacterial cellulose can be collected, rinsed in distilled water, and immersed in NaOH 1 N at 60°C for 90min to remove attached cells. The bacterial cellulose may then be washed in distilled water and dried at 50°C for 24h to evaluate the bacterial cellulose yield concentration in mg mL· ¹ ( ass( g) of BC/volume (ml_)) of culture medium).

Synthesis and secretion of cellulose

The synthesis of bacterial cellulose is a multistep process that involves two main mechanisms: the synthesis of uridine diphosphate (UDP-glucose), followed by the polymerisation of glucose into long and unbranched chains by cellulose synthase.

The proteins described herein are proteins that are involved in the synthesis and/or secretion of cellulose. In some embodiments, the microorganism or bacteria is modified to overexpress at least one, at least two, at least three, at least four, at least five, at least six, at least, seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve of the proteins involved in synthesis and/or secretion of cellulose.

The bacterial cellulose biosynthesis (bcs) operon encoding a cellulose synthase complex for cellulose biosynthesis and secretion was initially identified in Komagataeibacter xylinus (also known as Acetobacter xylinum and Gluconacetobacter xylinus). In some embodiments, the microorganism or bacterium is genetically modified to overexpress at least one protein from a cellulose synthase complex. In some embodiments, the microorganism or bacteria is modified to overexpress at least one, at least two, at least three, or at least four of the proteins from a cellulose synthase complex.

K. xylinus has been identified as the most efficient bacterial cellulose producer among cellulose producer species. Specifically bacterial cellulose produced by Komagataeibacter species, displays unique properties, including high mechanical strength, high water absorption capacity, high crystallinity, and an ultra-fine and highly pure fibre network structure (Vandamme, et al. 1998). Without being bound by theory, it is anticipated that genetic modification of a microorganism, bacterium or root-associated bacteria with the cellulose synthesis proteins of K. xylinus will result in an increased and more efficient production of cellulose.

In some embodiments, the genetically engineered microorganism, bacterium or root-associated bacterium is genetically modified with an exogenous nucleic acid that encodes at least one protein from a bacterial cellulose synthase complex. In some embodiments, the genetically engineered microorganism, bacterium or root-associated bacterium is modified with at least one protein from a cellulose synthase complex from K. xylinus. For the purposes of the invention, the components of the cellulose synthase complex are described herein.

For the purposes of this invention, “a bcs operon” encodes four protein subunits BcsA, BcsB, BcsC, and BcsD that form a cellulose synthase complex. The BcsA subunit, located on the cytoplasmic face of the inner membrane possesses a catalytic b-I, V^ ^IΐGqheίbGqeb domain responsible for polymerising monomers of uridine diphosphoglucose (UDP-glucose) into b-1,4- glucan chains of cellulose. The activity of the catalytic domain is regulated by the allosteric activator of bacterial cellulose synthesis, bis-(3'®5')-cyclic diguanylate. BcsB binds to BcsA in the periplasm by a single C-terminal transmembrane helix, where it stabilises BcsA and guides glucan chains through the periplasmic space using two carbohydrate-binding domains.

Secretion of bacterial cellulose from the periplasm to the extracellular environment is believed to be facilitated through the action of BcsC, which is predicted to form a pore in the outer membrane of K. xylinus based on its structure. Consistent with the view that BcsC is an outer membrane porin, is the observation that BcsC is essential for in vivo, but not in vitro bacterial cellulose synthesis. Finally, crystallisation of bacterial cellulose is achieved through the action of BcsD, a cylindrical octameric periplasmic protein that contains four spiral channels that facilitates hydrogen bonding of four glucan chains during export through BcsC. Furthermore, it has been demonstrated in K. xylinus that BcsC mutants were unable to produce cellulose fibrils, whereas BcsD mutants produced -40% less cellulose than the wild-type (Wong et al. 1990). Bacterial cellulose is distinguished from its plant equivalent by a high crystallinity index. Specifically, K. xylinus produces two crystalline allomorphs of bacterial cellulose known as cellulose I and cellulose II, which requires the cellulose synthase-associated BcsD subunit.

This subunit has been characterised as coupling cellulose polymerisation and crystallisation.

In some embodiments, the genetically engineered microorganism, bacterium, or root-associated bacterium of the invention is modified to overexpress at least one of the genes bcsA, bcsB, bcsC, and bcsD. In some embodiments, the genetically engineered microorganism, bacterium, or root-associated bacterium of the invention is modified to overexpress a bcsA gene and a bcsB gene. In some embodiments, the genetically engineered microorganism, bacterium, or root-associated bacterium of the invention is modified to overexpress a bcsA gene and a bcsB gene, and at least one of a bcsC gene and a bcsD gene. In further embodiments, the genetically engineered microorganism, bacterium, or root-associated bacterium of the invention is modified to overexpress at least bcsA, bcsB and bcsD. In some embodiments, the genetically engineered microorganism, bacterium, or root-associated bacterium of the invention is modified to overexpress a bcs operon. In further embodiments, the bcsA, bcsB, bcsC, bcsD, and bcs operon are each isolated from K. xylinus. cmcAx (also known as bcsZ) is located upstream of the bcs operon and encodes endo^-1,4- glucanase that has cellulose-hydrolysing ability. It has been demonstrated that in small amounts, exogenous CmcAx enhances bacterial cellulose production of K. xylinus, while endogenous overexpression of cmcAx increases bacterial cellulose yield. Without being bound by theory, it is anticipated that the cellulose hydrolysing activity of CmcAx may exert a regulatory effect on bacterial cellulose biosynthesis. In some embodiments, the genetically engineered microorganism, bacterium, or root-associated bacterium of the invention is modified to overexpress a cmcAx gene. In some embodiments, the microorganism is modified with a cmc gene. In further embodiments, the cmcAx gene is isolated from K. xylinus. ccpAx (also known as bcsH) is located in the same upstream operon as cmcAx, which encodes the cellulose-complementing protein (ccpAx) that is required for in vivo bacterial cellulose biosynthesis. CcpAx has been demonstrated to interact with BcsD in the periplasm. It is considered that this unique organisation might account for the extremely high activity of K.xylinus. In some embodiments, the genetically engineered microorganism, bacterium, or root- associated bacterium of the invention is modified to overexpress a ccpAx gene. In some embodiments, the microorganism is modified with a ccp gene. In further embodiments, the ccpAx gene is isolated from K. xylinus.

Downstream of the BC synthesis operon is bglAx (also known as bglxA ) encoding b- glucosidase, which is secreted and has the ability to hydrolyse more than three b-1, 4-glucose units (cellotriose). It has been demonstrated that whilst this enzyme is not essential for bacterial cellulose production, disruption of the bglAx gene causes a decrease in bacterial cellulose production (Tajima et ai, 2001; Kawano etai, 2002). In some embodiments, the genetically engineered microorganism, bacterium, or root-associated bacterium of the invention is modified to overexpress a bglAx gene. In some embodiments, the microorganism is modified with a bgl gene. In further embodiments, the bglAx gene is isolated from K. xylinus.

Phosphoglucomutase, also referred to as celB, is responsible for catalysing the interconversion between glucose- 1 -phosphate (G-1-P) and glucose-6-phosphate (G-6-P). Without being bound by theory, it is thought that the conversion of G-6-P to G-1-P facilitates the production of cellulose. Phosphoglucomutase has been demonstrated to be essential in the formation of extracellular cellulose, as pgm mutants are unable to produce cellulose. In some embodiments, the genetically engineered microorganism, bacterium, or root-associated bacterium of the invention is modified to overexpress a pgm gene. In further embodiments, the pgm gene is isolated from K. xylinus.

UTP-glucose-1-phoshate is an enzyme involved in carbohydrate metabolism, and synthesises UDP-glucose from glucose- 1 -phosphate (G-1-P) and UTP. UDP-glucose is a key component in the production of cellulose. In some embodiments, the genetically engineered microorganism, bacterium, or root-associated bacterium of the invention is modified to overexpress a galU gene. In further embodiments, the galU gene is isolated from K. xylinus. Diguanylate cyclase is an enzyme that catalyses 2 GTP into 2 diphosphate and cyclic GMP.

This may be introduced into a bacterial cell as a gene deg or as the edg operon. The edg operon comprises cyclic di-GMP phosphodiesterase ( pdeA ) and diguanylate cyclase (deg). Diguanylate cyclase, catalyses the formation of cyclic di-GMP and phosphodiesterase A catalyses the degradation. Without being bound by theory, cyclic di-GMP is considered to be an allosteric activator of bacterial cellulose synthesis. In some embodiments, the genetically engineered microorganism, bacterium, or root-associated bacterium of the invention is modified to overexpress a deg gene and/or a edg operon. In further embodiments, the deg gene and edg operon are each isolated from K. xylinus.

In some embodiments, the genetically engineered microorganism, bacterium, or root-associated bacterium is modified to overexpress at least one or more genes selected from the group comprising: a bcsA gene; a bcsB gene; a bcsC gene; a bcsD gene; a cmcAx gene; a ccpAx gene; a bglAx gene; a pgm gene; a galU gene; a edg operon; and a dgc gene.

In some embodiments the genetically engineered microorganism, bacterium, or root-associated bacterium is modified to overexpress the bcs operon and at least one of the following genes or operon: a) cmcAx gene; b) ccpAx gene; c) bglAx gene; d) pgm gene; e) galU gene; f) edg operon; and/or g) dgc gene.

In further embodiments, the genetically engineered microorganism, bacterium, or root- associated bacterium further comprises at least one, at least two, at least three, at least four, at least five, or at least six of the genes or operon as described by a) to g). In some embodiments, the genetically engineered microorganism, bacterium, or root-associated bacterium further comprises the genes and operon of a) to g). In some embodiments, the genetically engineered microorganism, bacterium, or root-associated bacterium further consists of the genes and operon of a) to g). In some embodiments, the genetically modified microorganism, bacterium, or root-associated bacteria of the invention comprise the bcs operon and at least the cmcAx gene, ccpAx gene, and bglAx gene. In some embodiments, the genetically modified microorganism, bacterium, or root-associated bacteria of the invention comprise the bcs operon and at least the cmc gene, ccp gene, and bgl gene. In some embodiments, the genetically modified microorganism, bacterium, or root-associated bacteria of the invention consist of the bcs operon and at least the cmcAx gene, ccpAx gene, and bglAx gene. In some embodiments, the genetically modified microorganism, bacterium, or root-associated bacteria of the invention consist of the bcs operon and at least the cmc gene, ccp gene, and bgl gene. In some embodiments, the genes are heterologous.

In some embodiments, a microorganism, bacterium, or root-associated bacterium comprising an endogenous bcs operon that does not comprise all of BcsA, BcsB, BcsC, and BcsD, may be modified with at least one of the genes of the bcs operon (bcsA, bcsB, bcsC, bcsD). Typically, the bacterium would be modified with a bcs gene which is does not normally express. For example, Pseudomonas fluorescens SBW25 expresses only BcsA, BcsB and BcsC of the bcs operon, and thus according to the invention would be modified to express BcsD. In some embodiments, the root-associated bacteria Pseudomonas fluorescens SBW25 is modified with an exogenous nucleic acid that comprises bcsD.

Without being bound by theory, it is anticipated that the bcs operon and any combination of the cmcAx gene, ccpAx gene, bglAx gene, pgm gene, the galU gene, the deg gene and/or the edg operon will facilitate the synthesis and secretion of cellulose in the host bacterium. In some embodiments, the genetically engineered microorganism, bacterium, or root-associated bacterium of the invention may comprise multiple copies of any of the genes or operons described herein.

In some aspects, the invention relates to the incorporation of cellulose synthesising genes (preferably cmcAX, ccpAX, bcsA, bcsB, bcsC, bscD, and/or bglxA) into a foreign host present in the soil. In some aspects, the invention provides a microorganism genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose, wherein the genetically modified bacterium is modified with one or more heterologous genes, wherein the genes comprise a bcsA gene, a bcsB gene, a bcsC gene, and/or a bcsD gene. In some embodiments, the genes comprise a bcsA gene, a bcsB gene, a bcsC gene, and a bcsD gene.

In some embodiments, the genes further comprise a cmcAx gene, a ccpAx gene, and/or a bglAx gene.

In some embodiments, the genetically modified bacterium is genetically modified with an exogenous nucleic acid comprising a bcs operon, wherein the bcs operon comprises a bcsA gene, a bcsB gene, a bcsC gene, and a bcsD gene. In some embodiments, the genetically modified bacterium is further modified with an exogenous nucleic acid comprising at least one of a cmcAx gene, a ccpAx gene, and a bglAx gene.

In some aspects, the invention provides a genetically engineered microorganism for producing cellulose, wherein the microorganism is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose, wherein the genetically modified bacterium is modified to overexpress at least one or more exogenous genes, wherein the exogenous genes are selected from the group comprising: a bcsA gene, a bcsB gene, a bcsC gene, a bcsD gene, and optionally a ccpAx gene. In some embodiments, the microorganism is a bacterium, optionally a root-associated bacterium. In some aspects, provided is a genetically engineered microorganism, comprising one or more heterologous genes coding for production of cellulose, wherein the genes are bcsA, bcsB, bcsC and/or bcsD. In some embodiments, the genes are bcsA, bcsB, bcsC and bcsD. In some embodiments, the genes further comprise a cmcAx gene, a ccpAx gene, and/or a bglAx gene. In some embodiments, the microorganism is a bacterium.

In further embodiments, the microorganism is a root-associated bacterium. In some embodiments, the genes are each isolated from K. xylinus.

In some embodiments, the genetically engineered microorganism further comprises a gene encoding green fluorescent protein (GFP). Without being bound by theory, providing a host cell that expresses GFP is considered to help with tracking the genetically modified microorganism, (e.g. bacteria) in the environment. Accordingly, in some embodiments, the genetically engineered microorganism, comprises one or more heterologous genes coding for production of cellulose, wherein the genes are bcsA, bcsB, bcsC and/or bcsD, and further comprises a gene encoding GFP.

Overexpression

The genetically engineered microorganism, bacterium, or root-associated bacterium of the invention is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose. In some embodiments, the genetically engineered microorganism, bacterium, or root-associated bacterium of the invention is modified to overexpress at least one protein from a cellulose synthase complex.

The term “overexpression” as used herein is where the protein(s) of interest is expressed in the microorganism or bacterium at a higher level than the level at which it is expressed in a reference microorganism or reference bacterium, respectively, optionally a comparable wild- type microorganism or bacterium, respectively, typically of the same strain or species. Overexpression may include but is not limited to constitutive or induced expression. In some embodiments, the microorganism or bacterium does not endogenously express the protein(s) of interest, any level of expression of that protein in the microorganism or bacteria cell is deemed an “overexpression” of that protein for purposes of the present invention. In the present invention, the terms “overexpression of at least one protein involved in cellulose synthesis and/or secretion” or “overexpression at least one protein from a cellulose synthase complex”, mean that the at least one of the proteins that are involved in the synthesis of cellulose is expressed in the microorganism or bacteria at a higher level than the level of which it is expressed in a comparable reference microorganism or bacterium, respectively. In some embodiments, the reference microorganism is a wild-type microorganism. In some embodiments, the reference microorganism is of the same species as the modified microorganism. The reference microorganism may be of the same strain as the modified microorganism. In some embodiments, the reference microorganism is a wild-type bacterium of the same strain or species as the modified microorganism. In some embodiments, the reference bacterium is a wild-type bacterium. In some embodiments, the reference bacterium is of the same species as the modified bacterium. The reference bacterium may be of the same strain as the modified bacterium. In some embodiments, the reference bacterium is a wild-type bacterium of the same strain or species as the modified bacterium.

Overexpression can be achieved in any way known to a skilled person in the art. In general, it can be achieved by increasing transcription/ translation of the gene, e.g. by increasing the copy number of the gene or altering or modifying regulatory sequences or sites associated with expression of a gene. For example, overexpression can be achieved by introducing one or more copies of the polynucleotide encoding the gene of interest operably linked to regulatory sequences (e.g. a promoter). The gene may be operably linked to a strong constitutive promoter and/or strong ubiquitous promoter in order to reach high expression levels. Such promoters can be endogenous promoters or recombinant promoters. Alternatively, it is possible to remove regulatory sequences such that expression becomes constitutive. One can substitute the native promoter of a given gene with a heterologous promoter which increases expression of the gene or leads to constitutive expression of the gene. Typically, genome editing methods such as CRISPR, TALENs, and Zinc Finger Nucleases can be used according to the invention to achieve overexpression of cellulose synthesis and/or secretion proteins. For example, CRISPR genome editing may be used to remove regulatory sequence(s), resulting in constitutive expression of the gene of interest. Cellulose synthesis and/or secretion proteins (e.g. proteins of the cellulose synthase complex and its associated proteins) may be overexpressed by more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more than 300% by the host cell compared to the host cell prior to engineering when cultured under the same conditions.

In one embodiment, overexpression of cellulose synthesis and secretion genes is achieved by altering or modifying regulatory sites associated with expression of a gene. In another embodiment, overexpression of cellulose synthesis and secretion genes is achieved by increasing the copy number of a cellulose synthesis and secretion gene. In a further embodiment, the microorganism, bacteria, or root-associated bacterium is modified with an exogenous nucleic acid comprising one or more cellulose synthesis and secretion genes. In some embodiments, the microorganism or bacteria are modified with one or more separate exogenous nucleic acids comprising one or more cellulose synthesis and secretion genes. In some embodiments, the exogenous nucleic acid is incorporated into a self-replicating plasmid within the microorganism or bacterium. In an alternative embodiment, the exogenous nucleic acid is incorporated into the genome of the microorganism or bacterium. In some embodiments, expression of the gene(s) of interest is transient. In some embodiments, expression of the gene(s) of interest is stable.

Detection of overexpression can be achieved in any way known to a skilled person in the art. Examples include, but are not limited to, detecting the proteins (machinery) for synthesis of cellulose e.g. cellulose synthase complex, by techniques such as Western Blot, qRT-PCT, and flow cytometry, or detecting the quantity of cellulose produced by the genetically engineered bacteria by techniques such as determining the weight of the dried and/or wet cellulose biomass.

In some embodiments, an exogenous nucleic acid is introduced into the microorganism, bacterium, or root-associated bacterium. In this specification, a nucleic acid may be any nucleic acid (DNA or RNA) having a nucleotide sequence having a specified degree of sequence identity to the genes of the bcs operon, a cmcAx gene; a ccpAx gene; a bglxA gene; a pgm gene; a galU gene; a cdg operon; and/or a dgc gene isolated from K. xylinus and to an RNA transcript of any one of these sequences, to a fragment of any one of the preceding sequences or to the complementary sequence of any one of these sequences or fragments. The specified degree of sequence identity may be from at least 60% to 100% sequence identity. More preferably, the specified degree of sequence identity may be one of at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In this specification, “an exogenous nucleic acid” refers to a nucleotide sequence that is foreign e.g. not endogenous to its host cell.

The term “endogenous” with reference to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in the host cell.

The term “heterologous” with reference to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in the host cell. In preferred embodiments, the exogenous nucleic acid is a heterologous nucleic acid.

The term “recombinant,” when used in reference to a subject cell, nucleic acid, protein or vector, indicates that the subject has been modified from its native state. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature. Recombinant nucleic acids differ from a native sequence by one or more nucleotides and/or are operably linked to heterologous sequences, e.g., a heterologous promoter in an expression vector. Recombinant proteins may differ from a native sequence by one or more amino acids and/or are fused with heterologous sequences. The engineered microorganism or bacterium may be considered a recombinant microorganism or bacterium. In one embodiment, the microorganism or bacterial cells are genetically engineered by introducing an expression cassette or vector comprising an exogenous nucleic acid sequence encoding the machinery for cellulose synthesis and secretion e.g. cellulose synthase complex into said cells. The nucleic acid sequence may be operably linked to one or more control sequences that direct the expression of said nucleic acid in the microorganism or bacteria cells. The control sequence may include a promoter that is recognised by the microorganism or bacterial cell. The promoter contains transcription control sequences that mediate the expression of the machinery for the synthesis of cellulose. The promoter may be any polynucleotide that shows transcription activity in the microorganism or bacterial cells including mutant, truncated, and hybrid promoters. The promoter may be a constitutive or inducible promoter, preferably a constitutive promoter. The control sequence may also include appropriate transcription initiation, termination, and enhancer sequences. In some embodiments, the expression cassette comprises, or consists of, a nucleic acid sequence that encodes the machinery for cellulose synthesis and secretion operably linked to a transcriptional promoter and a transcription terminator.

A “vector” as used herein is an oligonucleotide molecule (DNA or RNA) used as a vehicle to transfer foreign genetic material into a cell. The vector may be an expression vector for expression of the foreign genetic material in the cell. Such vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the gene sequence to be expressed. A vector may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be according to the invention. Suitable vectors include plasmids, binary vectors, viral vectors and artificial chromosomes (e.g. yeast artificial chromosomes). In some embodiments, the vector of the invention is an isolated vector. An “expression cassette” as used herein is a distinct component of vector DNA consisting of a gene and regulatory sequence to be expressed in a host cell. An expression cassette typically comprises one or more genes and the sequences controlling their expression.

As used herein, a “constitutive promoter” is a promoter which is active under most conditions and/or during most development stages. There are several advantages to using constitutive promoters in expression vectors used in biotechnology, such as: high level of production of proteins used to select transgenic cells or organisms; high level of expression of reporter proteins or scorable markers, allowing easy detection and quantification; high level of production of a transcription factor that is part of a regulatory transcription system; production of compounds that requires ubiquitous activity in the organism; and production of compounds that are required during all stages of development. Alternatively, a non-constitutive promoter can be used. As used herein, a "non-constitutive promoter" is a promoter which is active under certain conditions. In embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter is a sugar-induced promoter. In some embodiments, the promoter is an arabinose inducible promoter.

In preferred embodiments, the vector is a vector that when introduced into the microorganism or bacterial cell, is integrated into the genome and replicated together with the chromosome into which it has been integrated. In some embodiments, the integration of the genes encoding the machinery for cellulose synthesis will be integrated into the nonessential locus of a chromosome. A non-limiting example of a nonessential locus of a chromosome is locus -6-, on the 6.6Mbp chromosome of SBW25 (Rainey and Bailey, 1996) using the methodology shown by BAILEY et al., 1995 (which is incorporated by reference). Typically, insertion of the gene(s) of interest is mediated by site-directed homologous recombination. In some embodiments, insertion of the gene of interest is mediated by CRISPR genome editing. Typically, a CRISPR knock-in is mediated by homologous directed repair (HDR). Without being bound by theory, it is anticipated that extra metabolic activity from expressing novel gene sequences and environmental variability are safeguards against uncontrolled genetically modified bacteria multiplication in the environment. For example, a microorganism or bacterium that produces increased amounts of cellulose may only survive in environments that support its multiplication, such as growing in and around a plant root. If the genetically modified microorganism or bacteria grow in an unfavourable environment, the extra metabolic burden of producing increased amounts of cellulose will lead to reduced viability of the genetically modified microorganism or bacteria in the wider environment, thereby improving the safety of the genetically modified microorganism or bacteria.

A counter selectable marker may be used in the expression system. An example of selectable markers include the sucrose sensitivity system wherein the vector encodes sacB. Examples of suitable vectors include, but are not limited to, recombinant integrating or non-integrating vectors. Examples of vectors include pGEX series of vectors, pET series of vectors, and the pEX series of vectors. In some embodiments, the pEX18Ap vector is used. In some embodiments, a mini CTX1 vector is used. In some embodiments, a pFLP2 vector is used. A pFLP2 is an excision vector that can be used to remove unwanted sequence. In some embodiments, the insertion site in the host microorganism is attb defined by SEQ ID NO: 1: TGAGTTCGAATCTCACCGCCTCCGCCATAT.

In this specification the term “operably linked” may include the situation where a selected nucleotide sequence and regulatory nucleotide sequence are covalently linked in such a way as to place the expression of a nucleotide coding sequence under the influence or control of the regulatory sequence. Thus a regulatory sequence is operably linked to a selected nucleotide sequence if the regulatory sequence is capable of effecting transcription of a nucleotide coding sequence which forms part or all of the selected nucleotide sequence. Where appropriate, the resulting transcript may then be translated into a desired protein or polypeptide.

In some embodiments, the microorganism according to the invention is modified with a cmcAx gene, having the nucleic acid sequence as defined by SEQ ID NO: 2.

In some embodiments, the microorganism is modified with a cmcAx gene, having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% nucleic acid sequence identity to SEQ ID NO: 2.

In some embodiments, the microorganism according to the invention is modified with a ccpAx gene, having the nucleic acid sequence as defined by SEQ ID NO: 3.

In some embodiments, the microorganism is modified with a ccpAx gene, having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% nucleic acid sequence identity to SEQ ID NO: 3.

In some embodiments, the microorganism according to the invention is modified with a bcs operon, comprising a bcsA gene, a bcsB gene, a bcsC gene and a bcsD gene, having the nucleic acid sequence as defined by SEQ ID NO: 4.

In some embodiments, the microorganism is modified with a bcs operon, having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% nucleic acid sequence identity to SEQ ID NO: 4.

In some embodiments, the microorganism according to the invention is modified with a gfp gene having the nucleic acid sequence as defined by SEQ ID NO: 5.

In some embodiments, the microorganism is modified with a gfp gene, having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% nucleic acid sequence identity to SEQ ID NO: 5. In some embodiments, the microorganism according to the invention is modified with a bglAx gene having the nucleic acid sequence as defined by SEQ ID NO: 6.

In some embodiments, the microorganism is modified with a bglAx gene, having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% nucleic acid sequence identity to SEQ ID NO: 6.

In some embodiments, the microorganism is modified with a cmcAx gene having at least 65% nucleic acid sequence identity to SEQ ID NO: 2, a ccpAx gene having at least 65% nucleic acid sequence identity to SEQ ID NO: 3, a bcs operon having at least 65% nucleic acid sequence identity to SEQ ID NO: 4, a gfp gene having at least 65% nucleic acid sequence identity to SEQ ID NO: 5, and/or a bglAx gene having at least 65% nucleic acid sequence identity to SEQ ID NO: 6.

In some embodiments, the microorganism according to the invention is modified with a PhIZ quorum sensing promoter having the nucleic acid sequence as defined by SEQ ID NO: 7.

In some embodiments, the microorganism is modified with a PhIZ quorum sensing promoter, having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% nucleic acid sequence identity to SEQ ID NO: 7.

In some embodiments, the microorganism according to the invention is modified with a Rox quorum sensing promoter having the nucleic acid sequence as defined by SEQ ID NO: 8.

In some embodiments, the microorganism is modified with a Rox quorum sensing promoter, having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% nucleic acid sequence identity to SEQ ID NO: 8.

In some embodiments, the microorganism according to the invention is modified with a AfmR quorum sensing promoter having the nucleic acid sequence as defined by SEQ ID NO: 9.

In some embodiments, the microorganism is modified with a AfmR quorum sensing promoter, having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% nucleic acid sequence identity to SEQ ID NO: 9.

In some embodiments, the microorganism according to the invention is modified with a cmcAx gene having the nucleic acid sequence as defined by SEQ ID NO: 10.

In some embodiments, the microorganism is modified with a cmcAx gene, having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% nucleic acid sequence identity to SEQ ID NO: 10.

In some embodiments, the microorganism according to the invention is modified with a ccpAx gene having the nucleic acid sequence as defined by SEQ ID NO: 11.

In some embodiments, the microorganism is modified with a ccpAx gene, having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% nucleic acid sequence identity to SEQ ID NO: 11.

In some embodiments, the microorganism according to the invention is modified with a bcsA gene having the nucleic acid sequence as defined by SEQ ID NO: 12.

In some embodiments, the microorganism is modified with a bcsA gene, having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% nucleic acid sequence identity to SEQ ID NO: 12.

In some embodiments, the microorganism according to the invention is modified with a bcsB gene having the nucleic acid sequence as defined by SEQ ID NO: 13.

In some embodiments, the microorganism is modified with a bcsB gene, having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% nucleic acid sequence identity to SEQ ID NO: 13.

In some embodiments, the microorganism according to the invention is modified with a bcsC gene having the nucleic acid sequence as defined by SEQ ID NO: 14. In some embodiments, the microorganism is modified with a bcsC gene, having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% nucleic acid sequence identity to SEQ ID NO: 14.

In some embodiments, the microorganism according to the invention is modified with a bcsD gene having the nucleic acid sequence as defined by SEQ ID NO: 15.

In some embodiments, the microorganism is modified with a bcsD gene, having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% nucleic acid sequence identity to SEQ ID NO: 15.

In some embodiments, the microorganism according to the invention is modified with a gfp gene having the nucleic acid sequence as defined by SEQ ID NO: 16.

In some embodiments, the microorganism is modified with a gfp gene, having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% nucleic acid sequence identity to SEQ ID NO: 16.

In some embodiments, the microorganism according to the invention is modified with a bglAx gene having the nucleic acid sequence as defined by SEQ ID NO: 17.

In some embodiments, the microorganism is modified with a bglAx gene, having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% nucleic acid sequence identity to SEQ ID NO: 17.

In some embodiments, the microorganism is modified with a cmcAx gene having at least 65% nucleic acid sequence identity to SEQ ID NO: 10, a ccpAx gene having at least 65% nucleic acid sequence identity to SEQ ID NO: 11 , a bcsA gene having at least 65% nucleic acid sequence identity to SEQ ID NO: 12, a bcsB gene having at least 65% nucleic acid sequence identity to SEQ ID NO: 13, a bcsC gene having at least 65% nucleic acid sequence identity to SEQ ID NO: 14, a bcsD gene having at least 65% nucleic acid sequence identity to SEQ ID NO: 15, a gfp gene having at least 65% nucleic acid sequence identity to SEQ ID NO: 16, and/or a bglAx gene having at least 65% nucleic acid sequence identity to SEQ ID NO: 17. In further embodiments, a promoter having at least 65% nucleic acid sequence identity to SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, is operably linked to a cmcAx gene having at least 65% nucleic acid sequence identity to SEQ ID NO: 10, a ccpAx gene having at least 65% nucleic acid sequence identity to SEQ ID NO: 11 , a bcsA gene having at least 65% nucleic acid sequence identity to SEQ ID NO: 12, a bcsB gene having at least 65% nucleic acid sequence identity to SEQ ID NO: 13, a bcsC gene having at least 65% nucleic acid sequence identity to SEQ ID NO: 14, a bcsD gene having at least 65% nucleic acid sequence identity to SEQ ID NO: 15, a gfp gene having at least 65% nucleic acid sequence identity to SEQ ID NO: 16, and/or a bglAx gene having at least 65% nucleic acid sequence identity to SEQ ID NO: 17.

In some embodiments, the microorganism according to the invention is modified bySEQ ID NO: 18.

In some embodiments, the microorganism is modified with a nucleic acid having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% nucleic acid sequence identity to SEQ ID NO: 18.

The above sequences can be combined and used in any order.

Quorum sensing system

In some embodiments, expression of the cellulose synthesis genes is regulated by a cell- density quorum sensing promoter. In further embodiments, the expression of the cellulose synthesis genes is regulated by a cell-density quorum sensing system. In further embodiments, the quorum sensing system is under the control of a constitutive promoter. In further embodiments, the quorum sensing system regulates the promoter controlling expression of the genes disclosed herein. Without being bound by theory, it is anticipated that the use of a quorum sensing system controls the expression of the cellulose synthesis genes such that once the bacteria colonise the rhizosphere to a concentration threshold, the promoter is switched on and the cellulose synthesis will begin.

Quorum sensing (QS) is defined as the ability to detect and to respond to cell population density by gene regulation. As an example, bacteria can use quorum sensing to regulate phenotype expressions such as biofilm formation, virulence factor expression, motility, bioluminescence, nitrogen fixation, sporulation etc, which coordinate their behaviour. This function is based on the local density of the bacterial population in the immediate environment. In some embodiments, a quorum sensing operon is inserted into the host cell. In some examples, gram-positive bacteria use the autoinducing peptide (AIP) as an autoinducer, which acts as a signalling molecule. When a high-concentration of AIP is detected in the local environment, the AIP binds to a receptor to active a kinase. The kinase then phosphorylates a transcription factor, which then regulates transcription of gene(s). This is known as a two-component system. Thus, in some embodiments, a two-component system is used. In some embodiments, a two-component system comprises a sensor kinase (which detects the signalling molecule) and a response regulator (which regulates gene expression).

In another example, gram-negative bacteria produce N-acyl homoserine lactones (AHL) as a signalling molecule. Typically, these AHLs bind directly to transcription factors to regulate gene expression. In some embodiments, a one-step process is used. It is known that some gram negative bacteria also utilise a two-component system.

In some embodiments, the genes disclosed herein are regulated by a cell density-dependent auto-inducible promoter. In some embodiments, the cellulose synthesis and/or secretion genes disclosed herein are under the control of a cell-density quorum sensing promoter. It is anticipated that when the bacteria colonise the rhizosphere and reach a threshold density, the cellulose synthesising genes are switched on.

In some embodiments, the quorum sensing system comprises a gene encoding a sensor kinase and a gene encoding a response regulator. In further embodiments, the quorum sensing system further comprises a quorum sensing regulated promoter. In some embodiments, a nucleic acid comprising a gene encoding a sensor kinase and a gene encoding a response regulator is operably linked to a constitutive promoter. In further embodiments, a RoxS/RoxR quorum sensing system is used. In some embodiments, a RoxS/RoxR operon is inserted into the host cell. In alternative embodiments, the quorum sensing system comprises a gene encoding a signalling molecule (autoinducer) and a gene encoding a transcriptional/response regulator. In further embodiments, the quorum sensing system further comprises a quorum sensing regulated promoter. In some embodiments, a nucleic acid comprising a gene encoding a signalling molecule and a gene encoding a transcriptional/response regulator is operably linked to a constitutive promoter. In further embodiments, a PhzR/Phzl quorum sensing system is used. In some embodiments, a PhzR/Phzl operon is inserted into the host cell.

In some embodiments, the quorum sensing system activates the target gene promoter. In further embodiments, the response regulator binds to the target gene promoter.

QS-based auto-inducible promoter systems, specifically the RoxS/RoxR Quorum Sensing (QS) system of bacteria, is described in Meyers A, et al. 2019. The RoxS/RoxR quorum sensing system is a two-component system formed by a sensor histadine kinase (RoxS) and a response regulator (RoxR). It is anticipated that RoxS will result in the phosphorylation of RoxR, this phosphorylated RoxR will then regulate the expression of the cellulose synthesis and secretion genes disclosed herein, by binding to a putative RoxR recognition element. In some embodiments, a RoxS/RoxR quorum sensing system is used to control the expression of the cellulose synthesis and secretion genes. In some embodiments, a quorum sensing dependent RoxS/RoxR-promoter is used to control the expression of the cellulose synthesis and secretion genes. In some embodiments, a rox quorum sensing regulated promoter is used to control expression of the genes described herein. In some embodiments, a quorum sensing dependent RoxS/RosR-promoter is operably linked to a nucleic acid encoding the genes disclosed herein.

In some embodiments, the promoter comprises a RoxR recognition element.

Also described is the PhzR/Phzl quorum sensing system. This system comprises the transcriptional regulator PhzR and the AHL synthase Phzl. In some embodiments, a PhzR/Phzl quorum sensing system is used to control the expression of the cellulose synthesis and secretion genes. In some embodiments, a quorum sensing dependent PhzR/Phzl-promoter is used to control the expression of the cellulose synthesis and secretion genes. In some embodiments, a phz quorum sensing regulated promoter is used to control expression of the genes described herein. In some embodiments, a quorum sensing dependent PhzR/Phzl- promoter is operably linked to a nucleic acid encoding the genes disclosed herein.

In further embodiments, the quorum sensing system is under the control of a constitutive promoter. This can be seen in Figures 4 and 5.

In some embodiments, a RhlR/Rhll quorum sensing system is used to control the expression of the cellulose synthesis and secretion genes. In some embodiments, a RhlR/Rhll operon is inserted into the host cell. In the Rhll/R system, rhll directs the synthesis of N-(butanoyl)- homoserine lactone (C4-HSL), which then interacts with the cognate RhIR, influencing transcription of target genes. In some embodiments, a quorum sensing dependent RhlR/Rhll- promoter is used to control the expression of the cellulose synthesis and secretion genes. In some embodiments, a rhl quorum sensing regulated promoter is used to control expression of the genes described herein. In some embodiments, a quorum sensing dependent RhlR/Rhll- promoter is operably linked a nucleic acid encoding the genes disclosed herein.

In some embodiments, a Luxl/LuxR quorum sensing system is used to control the expression of the cellulose synthesis and secretion genes. In some embodiments, a Luxl/LuxR operon is inserted into the host cell. In some embodiments, a Lux/LuxR quorum sensing system is used to control the expression of the cellulose synthesis and secretion genes. In some embodiments, a quorum sensing dependent Luxl/LuxR-promoter is used to control the expression of the cellulose synthesis and secretion genes. In some embodiments, a lux quorum sensing regulated promoter is used to control expression of the genes described herein. In some embodiments, a quorum sensing dependent Luxl/LuxR-promoter is operably linked to a nucleic acid encoding the genes disclosed herein.

In some embodiments, a Afml/AfmR quorum sensing system is used to control the expression of the cellulose synthesis and secretion genes. In some embodiments, a Afml/AfmR operon is inserted into the host cell. In some embodiments, a Afml/AfmR quorum sensing system is used to control the expression of the cellulose synthesis and secretion genes. In some embodiments, a quorum sensing dependent Afml/AfmR- promoter is used to control the expression of the cellulose synthesis and secretion genes. In some embodiments, a afm quorum sensing regulated promoter is used to control expression of the genes described herein. In some embodiments, a quorum sensing dependent Afm l/AfmR-promoter is operably linked to a nucleic acid encoding the genes disclosed herein.

Without wishing to be bound by theory, the quorum sensing system acts as a biosafety element. The genetically engineered microorganisms of the invention are anticipated to colonise the rhizosphere environment of the plant of interest because the plant and bacterium live in a beneficial symbiotic relationship. In this biosafety system, the expression of cellulose may only be achieved when the concentration of bacteria is high. Therefore, when the genetically engineered microorganisms of the invention are not present in their optimal rhizosphere environment, the cellulose genes would not be expressed, and the genetically engineered microorganism would act as a wild-type strain.

In some embodiments, the heterologous cellulose synthesis and/or secretion genes are regulated by a quorum sensing system. In some embodiments, the heterologous cellulose synthesis and/or secretion genes are regulated by a quorum sensing regulated promoter.

In some embodiments, the quorum sensing system regulated promoter is operably linked to the nucleic acid encoding one or more of the exogenous genes of the invention, wherein expression of said genes is regulated by the quorum sensing system. In some embodiments, the exogenous genes comprise a bcsA gene; a bcsB gene; a bcsC gene; a bcsD gene; a cmc gene; a ccp gene; a bgl gene; a pgm gene; a galU gene; a cdg operon; and a dgc gene. In further embodiments, the genes a heterologous.

Compositions

In some embodiments, the genetically modified microorganism of the invention is delivered to plants as an inoculum that can be directly added to the soil. In some embodiments, the genetically modified bacterium of the invention is delivered to plants as a bacterial inoculum that can be directly added to the soil. In another embodiment, the genetically modified microorganism or bacterium of the invention is delivered to plants as a liquid formulation that can be directly added to the soil. In some embodiments, the microbial or bacterial inoculants are peat-based formulations. In further embodiments, the peat-based formulations are used to coat seeds or pellets for sowing in furrows. In some embodiments, the genetically modified microorganism or bacterium of the invention is delivered to plants in microbeads. In further embodiments, the microbeads are alginate microbeads. It is anticipated that these alginate microbeads encapsulate the microorganisms or bacteria and protect them against environmental stresses and release them into the soil gradually when soil microorganisms degrade the polymers.

Typically, the genetically modified microorganism or bacteria of the invention can be applied in combination with biofertilisers. A “biofertiliser” as used herein is a substance which contains living microorganisms which promotes plant growth by increasing the supply availability of primary nutrients to the host plant. Biofertilisers may add nutrients to the plant by nitrogen fixations, solubilising phosphorous, and stimulating plant growth through the synthesis of growth- promoting substances. Biofertilisers do not contain any chemicals which are harmful to the living soil. Examples include, Rhizobium, Azotobacter, Azospirilium and blue green algae (BGA). Additional examples include strains such as Pantoea agglomerans strain P5 or Pseudomonas putida strain P13, which are known in the art to solubilise phosphate from organic or inorganic phosphate sources. It is anticipated that the genetically modified microorganisms or bacteria of the invention can be used in combination with such biofertilisers. In some embodiments, the genetically modified microorganism or bacteria of the invention may administered to the soil in combination with a biofertiliser in a single composition. In some embodiments, the genetically modified microorganisms or bacteria of the invention may administered to the soil in combination with more than one biofertiliser in a single composition.

In another embodiment, the genetically modified microorganisms or bacteria of the invention are administered separately to the biofertiliser/ biofertilisers.

In some embodiments, the genetically modified microorganisms or bacterium of the invention is delivered to plants in combination with a fertiliser in a single composition. In some embodiments, the genetically modified microorganism or bacterium of the invention is delivered to plants in combination with more than one fertiliser in a single composition. In another embodiment, the genetically modified microorganism or bacterium of the invention is administered separately to the fertiliser/ fertilisers. As used herein a “fertiliser” is any material of natural or synthetic origin that are used to improve plant growth and yield.

In some embodiments, the composition of the invention is delivered to plants in microbeads. In further embodiments, the microbeads are alginate microbeads. Typically, alginate is the most common polymer material for the encapsulation of microorganisms for various industrial microbiological purposes, but other algal polysaccharides may be used (Bashan, Y., et al.

2002). The main advantages associated with alginate preparations are their non-toxic nature (reducing the impact to the local environment), degradation in the soil, their slow release of bacteria into the soil, and almost unlimited shelf life (Bashan Y et al. 2002). In some embodiments, the microbeads are applied as wet microbeads. In some embodiments, the microbeads are applied as dry microbeads. In some embodiments, the microbeads are between 100pm and 500pm in diameter. In a preferred embodiment, the microbeads are between 100pm and 200pm in diameter. It is anticipated that a microbead of between 100pm and 500pm in diameter will be able to hold >10 ⁶ CFU bead ¹ , which is sufficient to inoculate a seed. In some embodiments, the composition of the invention is delivered to plants as macrobeads. In further embodiments, the macrobeads are alginate macrobeads. It is anticipated that the alginate macrobeads behave in a similar manner to microbeads. In some embodiments, the macrobeads are between 1mm and 5mm in diameter. In a further embodiment, the macrobeads are between 1mm and 3mm in diameter.

In some embodiments, the microbial or bacterial composition is applied to a plant after planting but before harvest of said plant. In some embodiments, the microbial or bacterial composition is applied to the soil before planting a plant. In some embodiments, the plant is a crop plant. In some embodiments, the composition is used to coat seeds or pellets for sowing in furrows.

Without being bound by theory, it is anticipated that the genetically engineered microorganism, bacterium, or root-associated bacterium according to the invention will colonise the roots of plants following inoculation onto seeds and result in enhanced plant growth. The following steps outline the colonisation process: a) inoculation onto seed, b) multiplication in the spermosphere (region surrounding the seed) in response to seed exudates, c) attachment to the root surface, and d) colonisation of the developing root system.

In some embodiments, the microorganisms or bacteria are stored as a dried formula and delivered to soil as a liquid broth.

Methods

In one aspect of the invention, a method of increasing water-retention around plant roots is provided. The method comprising applying the genetically engineered microorganism, bacteria, or root-associated bacteria of the invention to the surrounding soil of the plant. In some embodiments, water-retention around plant roots is increased relative to the same plant(s) in the same conditions but without the microorganism of the invention. In some embodiments, the increase in water-retention can be measured by an increase in soil water content. Soil water content can be calculated on a gravimetric or volumetric basis. For example, gravimetric water content {dg) is the mass of dry soil, and is measured by weighing a soil sample {m _we i), drying the sample to remove the water, then weighing the dried soil sample (m^ _y ) using the following equation:

Alternatively, soil water content can be measured by volumetric water content (qn), which is the volume of liquid water per volume of soil. Volume is the ratio of mass to density (p), and can be calculated using the following equation:

In some embodiments, the soil water content is increased at least 2-fold, 3-fold, 4-fold, 5-fold, 6- fold and so on.

Another aspect of the invention provides a method of reducing water consumption in agriculture, comprising applying the genetically engineered microorganism, bacterium, or root-associated bacteria of the invention to the surrounding soil of the plant. In some embodiments, the reduction in water consumption in agriculture is reduced relative to the same plant(s) in the same conditions but without the microorganism of the invention. In some embodiments, the amount of water used in agriculture is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and so on.

Plants

It is anticipated that the genetically modified microorganism or bacteria of the invention will increase the amount of water retained around a plant root system and as a result increase crop viability and yield in climates that have reduced rainfall and/or drought. The increase in water retained is relative to the same plant in the same conditions (e.g. soil) without the bacteria of the invention.

In some embodiments of the invention, the plant is a cereal plant, a corn plant, a rice plant, a wheat plant, a soy plant, a sugarcane plant, a maize plant, a potato plant, a tomato plant, tobacco plant, and a cassava plant. In further embodiments, the plant is a cereal plant, a corn plant, a rice plant, a wheat plant, or a soy plant. An aspect of the invention provides a plant comprising the genetically engineered microorganism, bacterium, or root-associated bacteria of the invention. In some embodiments, the plant comprises the isolated genetically engineered root-associated bacterium of the invention, the population of genetically engineered root-associated bacteria of the invention, or the bacterial composition of the invention. In some embodiments, the genetically engineered root-associated bacterium, isolated genetically engineered root-associated bacterium, or the population of genetically engineered root-associated bacteria is associated with the plant roots. In some embodiments, the genetically engineered root-associated bacterium grows on the plant roots. In some embodiments, the genetically engineered microorganism, bacterium, or root- associated bacterium grows in the soil surrounding plant roots.

Carbon Capture

The invention provides a method of capturing carbon, comprising applying a genetically engineered microorganism to soil surrounding a plant root, wherein the microorganism is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose, and wherein the carbon is converted to cellulose by the microorganism.

In some embodiments, the genetically engineered microorganism is a microorganism according to the invention, an isolated genetically engineered microorganism of the invention, a population of genetically engineered microorganisms of the invention, or a composition of the invention.

In some embodiments, the carbon is absorbed as carbohydrates secreted from a plant and the carbohydrates are converted into cellulose by the microorganism. In some embodiments, production of cellulose results in increased water-retention around plant roots.

In some embodiments, the genetically engineered microorganism is selected from a bacterial cell, a fungal cell or an algae cell. In some embodiments the genetically engineered microorganism is a bacterium. In further embodiments, the genetically engineered microorganism is a root-associated bacterium.

The genetically engineered microorganism, bacterium or root-associated bacterium according to the invention excretes carbon-rich cellulose around plant roots. Besides storing large volumes of water, this secreted cellulose also sequesters a considerable amount of carbon, as explained below.

During the synthetic process, the glucose chains produced inside the microorganism or bacterial body extrude out through tiny pores present on their cell envelope. The glucose chains then form microfibrils that further aggregate to form cellulose ribbons. These ribbons generate a web-shaped network structure with plenty of empty spaces between the fibers. The well- separated nanofibrils of bacterial cellulose create an expanded surface area and highly porous matrix. The basic fibril structure consists of a b-1 4 glucan chain with the following molecular formula: (C6H10O5)n. The chains are held together by hydrogen bonds. Bacterial cellulose microfibrils are approximately 100-fold smaller than the fibrils of vegetal. The fibrous network of bacterial cellulose consists of well-arranged, three-dimensional nanofibers resulting in the formation of hydrogel film with a large surface area and considerable porosity. As it is not associated with lignin or hemicelluloses as in vegetal cellulose, bacterial cellulose is purer. Moreover, the three-dimensional nanofibril network has a high-water absorption capacity and tensile strength.

It is known that during plant photosynthesis atmospheric carbon dioxide is absorbed and converted to carbohydrates. These carbohydrates are then excreted in the exudate of the root. Such carbohydrates include many sugars comprising glucose, fructose, arabinose and others. These carbohydrates are considered to be essential as carbon sources for microbial life in the soil, driving cellular proliferation and growth. It is considered that roughly 30% of the sugars produced in a plant are secreted into the surrounding soil, which feeds the plant microbiome.

Natural bacteria found in the soil surrounding the plant does not provide a substantial sequestration event to slow/reduce climate change.

The inventor has surprisingly found that the genetically engineered microorganism, bacterium and/or root-associated bacteria according to the invention use this excreted glucose to grow, but also they are advantageously engineered to metabolise these sugars and excrete them as bacterial cellulose. Cellulose is composed of glucose molecules structurally formed with b-1,4- glycosidic bonds and intramolecular hydrogen bonds. This trapped carbon in the soil surrounding plant roots directly results in increased plant health (for example, by retaining water around plant roots). As a result, plants are able to absorb more carbon dioxide and the cycle goes on. As bacterial cellulose is able to degrade over a period of months, the sequestration of carbon can then be used by plants and microbes to grow stronger, maintaining the carbon in the soil. This will form a cyclical event that never escapes the soil.

In some embodiments, the increase in cellulose production results in increased plant viability. It is anticipated that this increase in plant viability results in increased atmospheric carbon capture by the plant and generation of sugars, which are converted to cellulose, thereby sequestering the carbon in the soil.

In some embodiments, cellulose production by the microorganism or bacterium results in increased carbon sequestration in the surrounding soil. In some embodiments, the carbon captured is increased at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold and so on. The increase in carbon captured is relative to the same plant(s) in the same conditions, but without the microorganism of the invention. In some embodiments, the carbon is atmospheric carbon.

In some embodiments, the carbohydrate secreted from a plant is a sugar. In further embodiments, the sugar is glucose, fructose, and/or arabinose.

Uses

In some aspects, the invention provides use of a genetically modified microorganism in agriculture, wherein the microorganism is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose.

In another aspect, the invention provides use of a genetically modified microorganism to increase water-retention around plant roots, wherein the microorganism is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose.

In another aspect, the invention provides use of a genetically modified microorganism in carbon capture, wherein the microorganism is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose, and wherein the carbon is converted into cellulose by the microorganism.

In some embodiments, the genetically engineered microorganism is a microorganism according to the invention, an isolated genetically engineered microorganism of the invention, a population of genetically engineered microorganisms of the invention, or a composition of the invention.

In some embodiments, the genetically engineered microorganism is selected from a bacterial cell, a fungal cell or an algae cell. In some embodiments the genetically engineered microorganism is a bacterium. In further embodiments, the genetically engineered microorganism is a root-associated bacterium.

In some embodiments, the microorganism is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose, wherein the genetically modified bacterium is modified with an exogenous nucleic acid comprising a bcs operon, wherein the bcs operon comprises a bcsA gene, a bcsB gene, a bcsC gene, and a bcsD gene. In further embodiments, the exogenous nucleic acid further comprises a ccpAx gene. In some embodiments, the exogenous nucleic acid further comprises a cmcAx gene, a ccpAx gene, and a bglAx gene. In further embodiments, the genes are heterologous. In some embodiments, the microorganism is genetically modified one or more heterologous genes, wherein the genes are selected from: a bcsA gene, a bcsB gene, a bcsC gene, a bcsD gene, a cmcAx gene, a ccpAx gene, and a bglAx gene. In some embodiments, the genes are each isolated from K. xylinus. In some embodiments, the genetically engineered microorganism is a bacterium, optionally a root-associated bacterium. In some embodiments, the genetically engineered microorganism is a plant growth- promoting rhizobacterium.

The features disclosed in the foregoing description, or in the following numbered embodiments or claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example +/- 10%.

Numbered embodiments

1. A genetically engineered microorganism for producing cellulose, wherein the microorganism is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose.

2. The genetically engineered microorganism of embodiment 1, wherein the cellulose is bacterial cellulose.

3. The genetically engineered microorganism of embodiment 1 or embodiment 2, wherein cellulose production is increased in the genetically modified microorganism compared to a reference microorganism.

4. The genetically engineered microorganism according to any one of the preceding embodiments, wherein the genetically modified microorganism is modified with an exogenous nucleic acid encoding at least one protein from a cellulose synthase complex.

5. The genetically engineered microorganism according to any one of the preceding embodiments, wherein exogenous nucleic acid comprises a bcs operon.

6. The genetically engineered microorganism according to embodiment 5, wherein the exogenous nucleic acid further comprises at least one of the following genes or operon: a) cmcAx gene; b) ccpAx gene; c) bglAx gene; d) pgm gene; e) galU gene; f) cdg operon; and/or g) dgc gene.

7. The genetically engineered microorganism according to embodiment 5, wherein the exogenous nucleic acid further comprises a cmcAx gene, a ccpAx gene, and a bglAx gene. 8. The genetically engineered microorganism according to any one of embodiments 5 to 7, wherein the bcs operon, cmcAx gene, ccpAx gene, bglAx gene, pgm gene, galU gene, cdg operon, and/or dgc gene are each isolated from K. xylinus.

9. The genetically engineered microorganism according to any one of the preceding embodiments, wherein the microorganism is a bacterium, optionally wherein the microorganism is Pseudomonas fluorescens.

10. The genetically engineered microorganism according to any one of the preceding embodiments, wherein the cellulose is secreted outside of the cell.

11. The genetically engineered microorganism according to embodiments 10, wherein the secreted cellulose forms a network outside of the cell.

12. The genetically engineered microorganism according to embodiments 11, wherein the secreted cellulose network increases water retention around plant roots.

13. The genetically engineered microorganism according to embodiments 12, wherein the plant is a cereal plant, a corn plant, a rice plant, a wheat plant, or a soy plant.

14. A method of increasing production of cellulose in a microorganism compared to a reference microorganism, wherein the method comprises a step of modifying the microorganism to overexpress at least one protein involved in synthesis and/or secretion of cellulose.

15. The method of increasing production of cellulose according to embodiments 14, wherein the microorganism is modified with an exogenous nucleic acid encoding at least one protein from a cellulose synthase complex.

16. A vector comprising an exogenous nucleic acid that comprises a bcs operon and at least one of a cmcAx gene, a ccpAx gene, a bglAx gene, a pgm gene, a galU gene, a cdg operon, and a dgc gene.

17. A method of producing a genetically engineered microorganism for producing cellulose, wherein the method comprises a step of modifying the microorganism to overexpress at least one protein involved in synthesis and/or secretion of cellulose comprising: a) isolating a microorganism; and b) introducing a vector comprising an exogenous nucleic acid comprising at least one of the genes selected from the group comprising: a bcsA gene; a bcsB gene; a bcsC gene; a bcsD gene; a cmcAx gene; a ccpAx gene; a bglAx gene; a pgm gene; a galU gene; a cdg operon; and a dgc gene, into the microorganism.

18. A genetically engineered microorganism obtainable by the method of embodiment 17.

19. An isolated genetically engineered microorganism according to any one of embodiments 1 to 13 or embodiment 18.

20. A population comprising the genetically engineered microorganism according to any one of embodiments 1 to 13 or embodiment 18.

21. A composition comprising the genetically engineered population of embodiment 20.

22. The composition according to embodiment 21, wherein the composition further comprises a fertiliser and/or a biofertiliser.

23. A method of increasing water-retention around plant roots, comprising applying a genetically engineered microorganism to soil surrounding a plant root, wherein the microorganism is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose.

24. The method according to embodiment 23, wherein the microorganism is selected from the genetically engineered microorganism according to any one of embodiments 1 to 13 or embodiment 18, the isolated genetically engineered microorganism of embodiment 19, the population of genetically engineered microorganisms of embodiment 20, or the composition of embodiments 21 or 22.

25. A method of reducing water consumption in agriculture, comprising applying a genetically engineered microorganism to soil surrounding a plant root, wherein the microorganism is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose.

26. The method according to embodiment 25, wherein the microorganism is selected from the genetically engineered microorganism according to any one of embodiments 1 to 13 or embodiment 18, the isolated genetically engineered microorganism of embodiment 19, the population of genetically engineered microorganisms of embodiment 20, or the composition of embodiments 21 or 22. A plant comprising the genetically engineered microorganism according to any one of embodiments 1 to 13 or embodiment 18, the isolated genetically engineered microorganism of embodiment 19, the population of genetically engineered microorganisms of embodiment 20, or the composition of embodiments 21 or 22, wherein the genetically engineered microorganism, isolated genetically engineered microorganism, or the population of genetically engineered microorganisms is associated with the plant roots. A method of capturing carbon, comprising applying a genetically engineered microorganism to soil surrounding a plant root, wherein the microorganism is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose, and wherein the carbon is converted to cellulose by the microorganism. The method according to embodiment 28, wherein the genetically engineered microorganism is a microorganism according to any one of embodiments 1 to 13 or embodiment 18, an isolated genetically engineered microorganism of embodiment 19, a population of genetically engineered microorganisms of embodiment 20, or a composition of embodiments 21 or 22. The method according to embodiment 28 or embodiment 29, wherein the carbon is absorbed as carbohydrates secreted from a plant and the carbohydrates are converted into cellulose by the microorganism. The method according to any one of embodiments 28 to 30, wherein production of cellulose results in increased water-retention around plant roots. Use of a genetically modified microorganism in agriculture, wherein the microorganism is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose. Use of a genetically modified microorganism to increase water-retention around plant roots, wherein the microorganism is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose. 34. Use of a genetically modified microorganism in carbon capture, wherein the microorganism is genetically modified to overexpress at least one protein involved in synthesis and/or secretion of cellulose, and wherein the carbon is converted into cellulose by the microorganism.

Examples

EXAMPLE 1 - Engineering of the root-associated bacteria

1. Bacterial strains and plasmids

Komagataeibacter xylinus DSM 2325 will be obtained from DSMZ (Braunschweig, Germany). Exemplary bacterial strains and plasmids are listed in Table 1.

K. xylinus is a member of the acetic acid bacteria, a group of Gram-negative aerobic bacteria that produce acetic acid during fermentation. K. xylinus is unusual among the group in also producing cellulose.

2. Gene manipulation

For genetic manipulation purposes, E. coli TOP10 cells will be used. E. coli cells will be cultivated in Luria-Bertani (LB) medium (Invitrogen, Carlsbad, CA) at 37°C with 225rpm orbital shaking. LB will be supplemented with antibiotics (50 pg/ml ampicillin) when needed for plasmid maintenance. All DNA manipulations will be conducted according to standard protocols (Sambrook, J., 2001).

Pseudomonas fluorescens strain SBW25 (Rainey and Bailey, 1996), will be utilised as a host for insertion of the bacterial biosynthetic cellulose machinery. A nonessential locus -6-, on the 6.6- Mbp chromosome of SBW25 will be chosen, as previously demonstrated (Rainey and Bailey, 1996) and the methodology shown by (BAILEY et al., 1995). Two fragments flanking the -6- locus (~200bp) will be amplified by conducting PCR with P. fluorescens genomic DNA; the genomic DNA will be prepared using genomic DNA extraction kit from Promega (Madison, Wl). The upstream and downstream flanking fragment will be amplified by PCR. The upstream and downstream regions of the -6- locus, bcs operon, pgm (phosphoglucomutase), galU (UTP- glucose-1 -phosphate), cdg operon, and dgc standalone gene (Table 2.) from K. xylinus (Jang et al., 2019) will be ligated into the pEX18Ap vector at the EcoRI restriction enzyme site using the In-fusion HD cloning kit (Clontech laboratories, Inc., mountain view, CA), resulting in the pEX- bcs vector. This plasmid will be transformed into E. coli TOP10 cells for the amplification and identification of the modified pEX-bcs plasmid. The purified plasmid will then be introduced into the -6- locus chromosomal site in P. fluorescens by electroporation, for the expression of the bcs bacterial biosynthetic cellulose machinery. 3. Growth conditions of engineered strains

The following conditions will be used for the visual verification of the successful bacterial biosynthetic cellulose machinery expression into P. fluorescens. Following this, greenhouse and field trials will be used for the optimisation of cellulose production in a model system. Nutrient broth media will be used for all cellulose synthesis production experiments using flasks containing: 3.0g/L meat extract, 10.0g/L peptone (enzymatic digest of casein), 5.0g/L sodium chloride, pH 7. Cells will be incubated for 5 days at 30°C under static conditions. The media will be supplemented with various carbohydrates for optimisation. Routine experimental optimisation of this protocol can be performed to adjust the specific parameters for best results according to particular field conditions.

Table 1. Description of the bacterial species and plasmids.

Table 2. Description of the genes essential for biosynthetic cellulose synthesis production in Komagataeibacter xylinus that will be used for the genetic modification in Pseudomonas fluorescens.

EXAMPLE 2 - Application of the genetically modified bacteria

The genetically modified bacteria will be delivered in biodegradable microbeads (microballs) containing a nutrient source which will be supplemented in currently commercially available biofertilisers.

In this example a method of inoculating plants (or seeds) with the genetically modified bacterium of the invention is described using alginate microbeads. These alginate microbeads encapsulate the bacteria and protect them against environmental stresses and release them into the soil gradually when soil microorganisms degrade the polymers. The raw material, kelp macroalga ( Macrocystis pyrifera), is a renewable marine resource of great abundance in the Pacific Ocean.

1. Micro bead formation

The microbeads may be produced using a device as described in Bashan et al. 2002, or any other suitable device. Typically, the microbeads produced will be around 100 to 200 pm in diameter. The bacteria of the invention will be cultured as described above and then the bacterial suspension will be mixed with 2% sodium alginate (CICIMAR, La Paz, Mexico), optionally skim milk without Ca may also be added to the alginate-bacterial suspension to produce beads that are more biodegradable. This suspension will then be pressurised at 10-15 psi using a commercial air compressor. Then the bacterial suspension will be forced to pass through a 222-pm-daimeter capillary exit, which will create a fine spray of miniature droplets. The mist will then be collected using a stainless steel flask rotating at 40rpm containing 0.1M CaCh to solidify the microbeads. The microbeads will then be allowed to cure in CaCh solution for 30mins. The wet microbeads will then be extracted from the CaCh solution, and then rinsed in 500ml saline solution (0.85% (w/v) NaCI) four times under aseptic conditions. Optionally the microbeads can be transferred into bacterial culture medium (in growth conditions) to allow for bacterial multiplication. The microbeads will then be separated from the suspension by filtration using Whatman filter paper, and rinsed three times with 500ml saline solution.

2. Drying procedures

Optionally, the microbeads may be dried before applying them to soil, plant roots, and/or seeds. In this drying method, 10g of microbeads can be placed as a thin layer on filter paper in a Petri dish and dried at 38±1°C for 48h. Then the dry microbeads can be collected in a hermetically sealed container with silica gel until they are used. Alternatively, dry microbeads may be prepared by standard lyophilisation.

The wet and/or dry microbeads comprising the genetically modified root-associated bacteria will then be applied to the soil, plant roots, and/or seeds.

EXAMPLE 3 - Engineering of the root-associated bacteria

1. Bacterial strains and plasmids

This method will use the Komagataeibacter xylinus CGMCC 2955 strain and the Mini CTX1 vector and the pFLP2 excision vector to remove unwanted sequences.

2. Gene manipulation

As before, for genetic manipulation purposes, E. coli TOP10 cells will be used. E. coli cells will be cultivated in Luria-Bertani (LB) medium (Invitrogen, Carlsbad, CA) at 37°C with 225rpm orbital shaking. LB will be supplemented with antibiotics (50 pg/ml ampicillin) when needed for plasmid maintenance. All DNA manipulations will be conducted according to standard protocols (Sambrook, J., 2001).

Pseudomonas strains CHAO, F113, FW300 N2E2 and Pf-5 will be utilised as a host for insertion of the bacterial biosynthetic cellulose machinery. The target insertion site in the genome of these strains is the attB site (SEQ ID NO 1: TGAGTTCGAATCTCACCGCCTCCGCCATAT). The cellulose synthesis genes (cmcAx, ccpAx, BcsA, BcsA, BcsC, BcsD, BglAx) will be inserted using a Mini CTX1 vector and a pFLP2 (Flp recombinase) vector. In this example, GFP will also be inserted as a reporter gene, this can be see in Figure 3. A quorum sensing operon can also be added to the Pseudomonas strains to regulate the promoter controlling cellulose synthase gene expression (see Figure 4). The quorum sensing operon, such as the Phzl/PhzR operon, will be under the control of a constitutive promoter.

3. Method Conjugations

Recipient Pseudomonas strains, as well as E. coli donor and helper strains, were grown in 3 ml LB (with antibiotic when appropriate) at 37°C with rolling for about 8 h. One milliliter of each culture was centrifuged at 8,000 ^c g for 2 min in microcentrifuge tubes. The culture supernatants were aspirated, cell pellets were resuspended in 1 ml LB, and cell suspensions were centrifuged. Aspiration, resuspension, and centrifugation were repeated. The supernatant was aspirated and cell pellets were resuspended in 35 pi LB. Cell suspensions were spotted onto LB agar and incubated at 37°C overnight. The cells were scraped off and resuspended in LB and serially diluted 10-fold, and 100 mI of each dilution was spread on Vogel-Bonner minimal medium (VBMM; 10 mM sodium citrate tribasic, 9.5 mM citric acid, 57 mM potassium phosphate dibasic, 17 mM sodium ammonium phosphate, 1 mM magnesium sulfate, 0.1 mM calcium chloride, pH 7.0) agar with antibiotic (gentamicin or tetracycline) and incubated at 37°C overnight. Chromosomal integration of miniTn7 was confirmed by PCR with oligonucleotide primers.

Electroporations

Recipient Pseudomonas strains will be grown in 3 ml LB in duplicate at 37°C with rolling for about 8 h. The two 3-ml cultures will then be pooled and dispensed into four microcentrifuge tubes. The cultures will be centrifuged at 8,000 ^c g for 2 min. Each cell pellet will then be resuspended in 1 ml 300 mM sucrose and centrifuged twice. The four cell pellets will then be resuspended and pooled in a total of 300 mI of 300 mM sucrose. One hundred microliters of each suspension will be transferred to 1-mm-gap-width electroporation cuvettes. One hundred nanograms of pFLP2 plasmid will be added to each suspension. Cells will be electroporated at 1,800 V in an Eppendorf electroporator 2510. Nine hundred microliters of LB will be added to each electroporation. Recovery cultures will then incubated at 37°C with rolling for 1 h. Cultures can be serially diluted 10-fold, spread on LB agar with antibiotic (carbenicillin), and incubated at 37°C overnight. Excision of antibiotic resistant cassette by Flp-FRT recombination Recipient Pseudomonas strains containing chromosomal gentamicin resistance cassette flanked by FRT recombination sites were electroporated with pFLP2 plasmid. Transformants were streaked on LB with carbenicillin, as well as on LB with gentamicin, to screen for excision of the gentamicin resistance cassette by Flp recombination. Gentamicin-sensitive transformants were streaked from LB with carbenicillin to LB with 5% sucrose. Strains that have the pFLP2 plasmid are sucrose sensitive, while those that have lost the plasmid are sucrose resistant. Sucrose-resistant colonies were streaked on LB, LB with gentamicin, and LB with carbenicillin to confirm both excision of the gentamicin resistance cassette and loss of the pFLP2 plasmid.

EXAMPLE 4 - Protocol for trial of genetically modified Pseudomonas fluorescens

1. Experimental design

Maize plants (cultivar Pioneer P7892) will be raised in 24 cell module trays, using seeds, and then similarly-sized plants will be transplanted at the 3-4 leaf stage into ~10L pots containing equal weight of commercially available sandy loam soil packed at the same bulk-density. Pots will be inoculated with one of nine inoculum treatments (Table 3). The control inoculum will consist of a mock inoculation using an equivalent volume of growth or other media lacking bacteria.

Five pot replicates will be used for each inoculum treatment. These will be arranged in five blocks (each of the nine treatments represented in each block) in a randomised block design on glasshouse benching, allowing data analysis to be conducted by Analysis of Variance (ANOVA). Environmental data will be captured in the glasshouse control system (temperature, relative humidity, solar radiation, set points).

Pots will be watered by hand daily according to the two treatments below, and will be fed twice a week with Hoagland’s solution once they have reached 8-10 leaves, or beginning when they have clearly exhausted the nutrients available in the pot. Feed solution will replace irrigation treatments given to reach field capacity, and volume of feed will be the same for all treatments, topped up with water to field capacity. Pest and diseases may be controlled with appropriate fungicides and insecticides.

To determine if the expected increase in soil water holding capacity will lead to an increase in plant growth, the trial will be carried out under conditions of limited water supply in a simulated rain-fed environment. Increased water holding capacity will not affect plant growth unless water is limiting, so a “well-watered” control treatment will be compared to a water-limited treatment: 1. Well-watered daily. Plants will be watered to field capacity every day or every other day so that water never becomes limiting to growth. No effect of inoculum is expected via the mechanism of increased soil water holding capacity when water is not limiting. Effects though other mechanisms may be observed.

2. Cycling between field capacity and growth-limiting soil water deficit. After plants have fully established, showing growth of an additional 2-3 leaves after transplanting, they will be watered to field capacity and then allowed to dry through transpirational water loss until control plants show signs of water deficit (wilting, leaf curling, reduced stomatal conductance compared to well-watered plants). They will then be watered back to field capacity, and the cycle repeated. Pots with greater water holding capacity will sustain growth longer before available water is depleted, and therefore will be expected to have longer periods of unrestricted growth (noting that increased growth rate will also increase transpiration, so eventually increased growth will balance out the increased water holding capacity).

Pots will be spaced at 30 cm spacings in two rows arranged on 1 m wide benches. The glasshouse compartment will be heated to 25/20°C day/night and supplementary illumination will be provided by high-pressure sodium lamps on a 16/8 hour day/night cycle. Maize has optimal growth at 25°C (broad optimum between 21 and 27°C).

Five replicate pots will be used for all treatments except for “No Pseudomonas” treatment where 10 replicates will be used (as all lines will be compared to this baseline). There will be two irrigation treatments.

Total number of pots = [(5 reps x 8 inocula) + (10 reps x 1 inoculum)] x 2 treatments = 100

Bench area = 1 x 6 m ² and 1 x 9 m ² (30 cm x 100, two rows on a 1 m wide bench, split over two benches containing either 2 or 3 “blocks”). Guard plants will be installed at the ends of each row (total of 8 plants) to reduce edge effects.

Table 3: Inoculum treatments · · 1

2. Measurements

Water holding capacity:

Water holding capacity will be measured with a funnel technique in which soil and roots from pots is macerated to mix soil and roots, sampled to estimate gravimetric water content (weigh before and after drying), and then packed into a funnel. Water will be added to the top of the funnel and the amount of water retained will be recorded based on the mass of water applied and draining through the funnel. This is a rapid method that will allow the total water holding capacity between field capacity (water held after free drainage) and oven-dried soil to be calculated. This method includes the water between permanent wilting point and oven dryness which is considered too tightly bound to the soil to be available to the plant. Alternatively, a moisture release curve which is a plot of soil water potential (in MegaPascals, MPa) against gravimetric water content (g g ^_1 ) can be calculated. The latter method allows calculation of the gravimetric water held between soil at field capacity (-0.01 MPa) and permanent wilting point (- 1.4 MPa soil water potential), i.e. only the water available to the plant. One measurement per pot = 100.

The funnel method will be validated as being responsive to soil cellulose content by using soil mixed with varying amounts of cellulose powder purchased from Fisher Scientific. Approximately 20 measurements.

Microbial colonisation of the rhizosphere:

A ~10g sample of macerated soil/roots from the water capacity experiment (one sample per pot at harvest) will be mixed with water or buffer solution, and then filtered. Serial dilutions will be plated onto the appropriate selective media, and then incubated for 48 hrs at 25°C. Colony forming units (cfus) will be counted and cfu ml ¹ calculated. A positive control would consist of inoculum applied to a sample of non-inoculated soil immediately prior to extraction (done at time of pot inoculation). Three replicates per treatment and three serial dilutions plus controls = (3 reps x 9 inocula x 2 irrigation treatments x 3 dilutions) + (8 control inoculations x 3 replicates x 3 dilutions) = 162 +

72 = 234 plates.

Carbon content of the soil:

A ~20g sample of soil will be taken from the rhizosphere at harvest (prior to macerating soil/roots for the water capacity experiment), and any root fragments will be removed by sieving at 2mm. Total organic carbon (TOC) will be measured by elemental analyser following hydrochloric acid treatment to remove carbonates.

One sample per pot = 100 samples. Positive control analysis will also be conducted on soil mixed with cellulose (produced in the water capacity experiment) = 20 samples.

Plant health: a) During plant growth, plant height and leaf number will be measured at weekly intervals using methods defined by agronomy practices.

Briefly, height will be defined as “from the soil surface to the highest point of the arch of the uppermost leaf whose tip is pointing down”. Leaf number will be recorded using three rapid methods:

1. Number of leaf tips that have emerged from the whorl.

2. Number of leaves, starting from the lowest leaf and finishing with the last leaf that has arched over (leaf tip pointing down).

3. Number of leaves with visible collars (i.e. leaves emerged from the whorl). b) Leaf chlorophyll content will be measured with a leaf chlorophyll meter (CCM-200) at weekly intervals on a standard leaf (e.g. uppermost arched leaf, two readings per leaf) validated with a standard curve (acetone extraction and spectrophotometer). 3 weeks x 200 measurements = 600. c) At plant harvest, the total aboveground matter will be chopped, bagged and dried at 80°C until fully dried. Dry weights per plant will be recorded (100 measurements).

Plant mineral content may also be measured using standard techniques. References

A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

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